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|HAI LALA AT MATALAMITAKAUS009816096B2 HUOLEHT I (12 ) United States Patent (10 ) Patent No. : US 9 ,816 , 096 B2 Heintz et al. (45 ) Date of Patent: Nov . 14 , 2017

(54 ) METHODS AND COMPOSITIONS FOR 6 , 143 , 566 A 11/ 2000 Heintz et al. TRANSLATIONAL PROFILING AND 6 , 156 , 574 A 12 / 2000 Heintz et al. 6 , 252 , 130 B1 6 / 2001 Federoff MOLECULAR PHENOTYPING 6 , 270, 969 B1 8 / 2001 Hartley et al. 6 , 403 ,374 B1 6 / 2002 Tsien et al. (71 ) Applicant: THE ROCKEFELLER 6 , 410 , 317 B1 6 /2002 Farmer UNIVERSITY, New York , NY (US ) 6 , 441 , 269 B1 8 / 2002 Serafini et al . 6 , 485 , 912 B1 11/ 2002 Heintz et al. @ 6 ,495 ,318 B2 12 / 2002 Harney (72 ) Inventors: Nathaniel Heintz , Pelham Manor, NY 6 ,635 ,422 B2 10 / 2003 Keene et al. (US ); Paul Greengard , New York , NY 6 , 821, 759 B1 11/ 2004 Heintz et al . (US ); Myriam Heiman , New York , NY 7 , 098, 031 B2B2 8 /2006 Choulika et al . (US ) ; Anne Schaefer , New York , NY 7 ,297 ,482 B2 11 /2007 Anderson et al . (US ) ; Joseph P . Doyle , New York , NY 7 , 393 , 632 B2 7 / 2008 Cheo et al. 2003 /0119104 A1 6 /2003 Perkins et al. (US ); Joseph D . Dougherty , St. Louis , 2004 / 0023256 A1 2 / 2004 Puglisi et al . MO (US ) 2005 / 0009028 Al 1 /2005 Heintz et al. 2006 /0183147 AL 8 /2006 Meyer - Franke (73 ) Assignee : THE ROCKEFELLER 2011/ 0314565 Al 12 /2011 Heintz et al . UNIVERSITY , New York , NY (US ) FOREIGN PATENT DOCUMENTS ( * ) Notice : Subject to any disclaimer , the term of this patent is extended or adjusted under 35 EP 1132479 A1 9 / 2001 WO WO -01 / 48480 A1 7 /2001 U . S .C . 154 (b ) by 0 days . WO WO - 01 / 58954 A2 8 / 2001 WO WO -01 / 58954 A3 3 / 2002 (21 ) Appl. No. : 14 /553 , 937 WO WO -02 /064749 A2 8 / 2002 WO WO - 2002/ 064749 A3 3 /2003 ( 22 ) Filed : Nov . 25 , 2014 WO WO -03 / 064604 A2 8 / 2003 WO WO -03 /064604 A3 11/ 2003 (65 ) Prior Publication Data US 2015 /0082470 A1 Mar. 19 , 2015 OTHER PUBLICATIONS Agafonov , et al. , Proteins on ribosome surface : measurements of Related U .S . Application Data protein exposure by hot tritium bombardment technique, Proceed (63 ) Continuation of application No. 12 / 918 ,761 , filed as ings of the National Academy of Science U S A , 94 ( 24 ): 12892 application No . PCT/ US2009 /001614 on Mar . 12 , 12897 ( 1997 ). 2009 , now abandoned . Anthony , et al ., Creating somatic cell genetic mosaics in the mouse , Cell , 121 ( 3 ) : 322 - 323 (2005 ) . (60 ) Provisional application No. 61 / 070 ,327 , filed on Mar . Antic , et al. , ELAV tumor antigen , Hel- N1 , increases translation of 21, 2008 , provisional application No . 61/ 036 , 049 , neurofilamentMmRNA and induces formation of neurites in human teratocarcinoma cells, Genes Development, 13 ( 4 ): 449 -461 (1999 ). filed on Mar. 12, 2008 , provisional application No . Ashiya , et al . , A neuron - specific splicing switch mediated by an 61 / 199 , 108 , filed on Nov. 12 , 2008 , provisional array of pre- mRNA repressor sites : evidence of a regulatory role for application No . 61 /036 ,058 , filed on Mar. 12 , 2008 . the polypyrimidine tract binding protein and a brain -specific PTB counterpart, RNA , 3 ( 9 ): 996 - 1015 ( 1997 ) . (51 ) Int. Cl. Brodersen , et al . , The social life of ribosomal proteins , FEBS CI2N 15 /62 ( 2006 .01 ) Journal , 272 ( 9 ) : 2098 -2108 (2005 ) . C12N 15 / 11 ( 2006 .01 ) Broude , et al. , Proteins of the 30 - S subunit of Escherichia coli GOIN 33 /50 ( 2006 . 01 ) ribosomes which interact directly with natural mRNA , European A01K 67 /027 ( 2006 .01 ) Journal of Biochemistry , 132 ( 1 ) : 139 - 45 ( 1983 ) . ( 52 ) U .S . CI. Buskila , et al ., Serum monoclonal antibodies derived from patients CPC ...... C12N 15 /62 ( 2013 . 01 ) ; A01K 67 / 0278 with multiple myeloma react with mycobacterial phosphoinositides ( 2013 .01 ); C12N 15 / 111 (2013 .01 ); GOIN and nuclear antigens, Clin Exp Immunol, 76 ( 3 ): 378 -83 ( 1989 ). 33 /5023 (2013 .01 ) ; GOIN 33 / 5058 ( 2013 .01 ); A01K 2217 / 206 ( 2013 .01 ) ; A01K 2227 / 105 (Continued ) (2013 .01 ) ; A01K 2267 /01 (2013 .01 ) ; C12N Primary Examiner — Channing S Mahatan 2320 / 12 ( 2013 .01 ) ; GOIN 2800 /285 ( 2013 .01 ) (74 ) Attorney, Agent, or Firm — Choate , Hall & Stewart (58 ) Field of Classification Search None LLP ; Rober N . Sahr ; David E . Shore See application file for complete search history. (57 ) ABSTRACT (56 ) References Cited Methods and compositions are provided for translational profiling and molecular phenotyping of specific tissues, cells U . S . PATENT DOCUMENTS and cell subtypes of interest . The methods provided herein 5, 075 ,227 A 12 / 1991 Hagen facilitate the analysis of gene expression in the selected 5 , 795 , 723 A 8 / 1998 Tapscott et al. subset present within a heterogeneous sample . 6 , 110 ,711 A 8/ 2000 Serafini et al . 6 , 130 ,090 A 10 / 2000 Heintz et al . 18 Claims, 54 Drawing Sheets US 9, 816 ,096 B2 Page 2

References Cited Kubota , et al. Nuclear and nucleolar targeting of human ribosomal ( 56 ) protein S25 : ; common features shared with HIV - 1 regulatory pro teins , Oncogene , 18 ( 7 ) : 1503 - 1514 ( 1999 ) . OTHER PUBLICATIONS Lalanne , et al . Complete sequence of mouse S6 ribosomal protein , Nucleic Acids Research , 15 ( 12 ) :4990 ( 1987) . Ceman , et al. , Isolation of an FMRP -associated messenger Lee , et al. , Cocaine - induced dendritic spine formation in D1 and D2 ribonucleoprotein particle and identification of nucleolin and the dopamine receptor- containing medium spiny neurons in nucleus fragile X - related proteins as components of the complex , Molecular accumbens, Proceedings of the National Academy of Science, USA , Cell Biology , 19 ( 12 ) : 7925 - 7932 ( 1999 ) . 103 ( 9 ) : 3399 - 3404 (2006 ) . Chaible , et al . , Genetically -modified animals for use in research and Lerner and Steitz , Antibodies to small nuclear RNAs complexed biotechnology, Genetic Molecular Research , 9 ( 3 ) : 1469 - 1482 with proteins are produced by patients with systemic lupus erythe ( 2010 ) . matosus , Proceedings of the National Academy of Science USA , Chambers , et al. Translational regulation of hepatic HMG - COA 76 ( 11 ) : 5495 - 9 ( 1979 ) . reductase by dietary cholesterol, Biochem Biophys Res Commun , Liang , et al. , An estrogen -dependent polysomal protein binds to the 232( 2 ): 278 -281 ( 1997 ) . 5 ' untranslated region of the chicken vitellogenin mRNA , Nucleic Christopher, et al. Implications of N and C - terminal proximity for Acids Research , 19 ( 9 ): 2289 - 2294 ( 1991) . protein folding, Journal of Molecular Biology , 257 (1 ): 175 -87 Lin , et al. , The primary structure of rat liver ribosomal protein L37 . ( 1996 ) . Homology with yeast and bacterial ribosomal proteins , Journal of Chu , et al. , Identification of a thymidylate synthase Biologic Chemistry, 258 ( 17 ) : 10664 - 10671 ( 1983 ) . ribonucleoprotein complex in human colon cancer cells , Molecular Misulovin , et al. , A rapid method for targeted modification and Cell Biology , 14 ( 1 ) :207 -213 ( 1994 ) . screening of recombinant bacterial artificial chromosome, Journal Chu , et al. , Identification of in vivo target RNA sequences bound by of Immunology Methods, 257 ( 1- 2 ): 99 - 105 (2001 ) . thymidylate synthase, Nucleic Acids Research , 24 ( 16 ): 3222 -3228 Nevskaya et al. , Ribosomal protein LI recognizes the same specific ( 1996 ). structural motif in its target sites on the autoregulatory mRNA and Copeland , et al . A novel RNA binding protein , SBP2, is required for 23S rRNA , Nucleic Acids Research , 33 ( 2 ): 478 -485 (2005 ). the translation of mammalian selenoprotein mRNAs, EMBO Jour Noain et al. , Identification of brain neurons expressing the nal, 19 ( 2 ): 306 - 314 ( 2000 ) . dopamine D4 receptor gene using BAC transgenic mice , European De Jonge et al. , Transcriptional profile of the human peripheral Journal of Neuroscience , 24 ( 9 ) : 2429 - 2438 ( 2006 ) . nervous system by serial analysis of gene expression , Genomics, Office Action for U . S . Appl. No . 10 /494 , 248 , (dated Dec . 23 , 2010 ) , 82 ( 2 ) : 97 - 108 (2003 ) . 14 pages . Doyle , et al . , Application of a translational profiling approach for Office Action for U . S . Appl. No . 10 / 494 , 248 , (dated Feb . 19 , 2010 ) , the comparative analysis of CNS cell types , Cell, ( 2008 ) . 21 pages. European Search Report and Opinion for EP Application No . Office Action for U . S . Appl. No . 10 /494 ,248 , (dated Jul . 29, 2008 ) , 09719684 . 4 ( dated Feb . 21 , 2012 ) . 27 pages . Fisher , et al. , Pulse labeling of small nuclear ribonucleoproteins in Office Action for U . S . Appl. No . 10 /494 ,248 , (dated Jun . 5, 2009 ) , vivo reveals distinct patterns of antigen recognition by human 20 pages. autoimmune antibodies , The Proceedings of the National Academy Office Action for U . S . Appl. No. 10 /494 , 248 ( dated Sep . 25 , 2007 ) , of Science US A . , 81( 10 ) : 3185 - 3189 ( 1984 ) . 13 pages . Gerfen , et al. , D1 and D2 dopamine receptor -regulated gene expres Office Action for U . S. Appl. No . 12 /918 ,761 , 12 pages (dated Aug . sion of striatonigral and striatopallidal neurons, Science, 250 (4986 ): 26 , 2014 ) , 10 pages. 1429 - 1432 ( 1990 ) . Office Action for U .S . Appl. No . 13/ 104, 316 (dated Jul. 26 , 2012 ), Gimautdinova , et al. , The proteins of the messenger RNA binding 14 pages. site of Escherichia coli ribosomes, Nucleic Acids Research , Peng, et al. , RNA stabilization by the Au - rich element binding 9 ( 14 ) : 3465 - 3481 ( 1981) . protein , HuR , an ELAV protein , The EMBO Journal , 17 ( 12 ): 3461 Gong , et al. , A gene expression atlas of the central nervous system 3470 ( 1998 ) . based on bacterial artificial chromosomes . Nature, 425 (6961 ) :917 Remacha et al. , Proteins PI, P2 , and PO , components of the 925 (2003 ) . eukaryotic ribosome stalk . New structural and functional aspects , Gong , et al ., Targeting Cre recombinase to specific neuron popula Biochem Cell Biology , 73 ( 11 - 12 ) : 959 - 968 ( 1995 ) . tions with bacterial artificial chromosome constructs , Journal of Ristevski, S . , Making better transgenic models: conditional, tem Neuroscience , 27 ( 37 ) :9817 - 23 (2007 ) . poral , and spatial approaches, Mal Biatechnol, 29 ( 2 ) : 153 -63 ( 2005 ) . Gonzalo and Reboud , The puzzling lateral flexible stalk of the Roche , et al . , SsrA -mediated peptide tagging caused by rare codons ribosome, Biology Cell, 95 ( 3 - 4 ) : 179 - 93 ( 2003 ) . and tRNA scarcity , The EMBO Journal , 18 ( 16 ) :4579 - 4589 ( 1999 ) . Hagen -Mann et al ., RT- PCR and alternative methods to PCR for in Rusk , N ., Targeted translational profiling , Nature Methods, 6 ( 1) : 7 vitro amplification of nucleic acids, Exp Clin Endocrinol Diabetes , ( 2009 ) . 103 ( 3 ) : 150 - 155 ( 1995 ) . Sano , et al ., Streptavidin - containing chimeric proteins: design and Heiman , et al. A translational profiling approach for the molecular production , Methods Enzymol, 326 : 305 -311 (2000 ). characterization of CNS cell types, Cell (2008 ) . Sanz , et al ., Cell - type - specific isolation of ribosome- associated Heintz , N . , Analysis of mammalian central nervous system gene mRNA from complex tissues , The Proceedings of the National expression and function using bacterial artificial chromosome Academy of Science USA , 106 (33 ) :13939 - 13944 (2009 ). mediated transgenesis , Human Molecular Genetics, 9( 6 ): 937 - 943 Schena , et al. , Parallel human genome analysis : microarray- based ( 2000 ) . expression monitoring of 1000 genes , The Proceedings of the Heintz , N ., BAC to the future: the use of bac transgenic mice for National Academy of Science USA , 93 ( 20 ) : 10614 - 10619 ( 1996 ) . neuroscience research , Nat Rev Neuroscience, 2 ( 12 ): 861- 70 (2001 ). Smith , K . 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US 9 ,816 ,096 B2 Page 3

( 56 ) References Cited OTHER PUBLICATIONS Trifillis , et al. , Finding the right RNA : identification of cellular mRNA substrates for RNA - binding proteins, RNA , 5 ( 8 ) : 1071 -82 (1999 ). Uchiumi and Kominami, Binding of mammalian ribosomal protein complex PO .P1 . P2 and protein L12 to the GTPase - associated domain of 28 S ribosomal RNA and effect on the accessibility to anti - 28 S RNA autoantibody, Journal of Biol Chemistry, 272 ( 6 ) : 3302 - 8 ( 1997 ) . Walles -Granberg , et al ., Ribosomes with large synthetic N - terminal extensions of protein S15 are active in vivo , Biochim Byophys Acta , 1544 ( 1 - 2 ) : 378 - 385 (2001 ) . Wilson , et al. , Ribosomal proteins in the spotlight , Crit Rev Biochem Mol Biol, 40 ( 5 ) : 243 -67 ( 2005 ) . Yang , et al. BAC -mediated gene -dosage analysis reveals a role for Ziprol (Ru49 /Zfp38 ) in progenitor cell proliferation in cerebellum and skin , Nat Genet , 22 ( 4 ): 327 - 35 ( 1999 ) . Yang , et al. , Homologous recombination based modification in Escherichia coli and germline transmission in transgenic mice of a bacterial artificial chromosome, Nature Biotechnology, 15 (9 ) :859 65 ( 1997 ) . Zong , et al. Messenger RNA translation state : The second dimen sion of high - throughput expression screening , PNAS , 96 ( 19 ) : 10632 - 10636 ( 1999 ) . U . S . Patent Nov . 14, 2017 Sheet 1 of 54 US 9 ,816 ,096 B2

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Figure 22

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Figure 22 continued Acetylcholine Receptors

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nicotinic , a polypeptide 5 * nicotinic , a polypeptide 6 chmar 04 e9 Mimi nicoinic , B polypeptide 1 Chmba 07 nicoinic , B polypeptide 2

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Figure 22 continued Selected novel and expressed receptors Gene ip /ub 1 Title Acvr1 1 . 24 Activin A type 1

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Ghr 0 .82 Growth hormonennnn. ' . Gor3 2 . 48 G' -protein ' ' ' ' ' coupled 3 Gor22 1 . 3 G protein - coupled 19 Gpr270 . 49 G protein.. -.coupled ...... 27 Gpr45 G - protein coupled 45 Gor49 06 G -protein coupled 49 Gor54 G - protein' - ' - ' - ' - ' - ' . ' ' - ' - ' - ' - ' - ' - ' - ' - ' - ' - ' - ' - ' - coupled' - ' - ' - ' . ' . ' - ' - ' - ' - ' - ' - ' - - - ' - ' - ' - ' - ' - ' - ' - ' . ' . ' . 54 Gpr61 109 G - protein coupled 61 Gpr68 167 G protein - coupled 68. * . * . * ...... * . . * . *. * . . * . * . Gor831 . 34 G - protein coupled 83 Gpr85 0 . 85 G protein - coupled 85 Gor123 2 .48 G protein - coupled 123 Gpr135 0 .47 G proteinwww - coupled 135 Gpr139 0 .51 G protein - coupled 139 Ifngr 0 . 72 Interferon gamma Ifngr2 2 . 8 Interferon gamma 2 Oprs12 .66 Opioid sigma 1 Tnfrsf12 : 2 .62 TNFR superfamily , 12awww5 Tnfrsf1a 1 .44 TNFR superfamily , 1a

Tnfrsf21 1. 68 TNFR superfamily...... iii i . i . i . i . , 21 Vdr 8 .54 Vitamin D U . S . Patent Nov. 14 , 2017 Sheet 35 of 54 US 9 ,816 ,096 B2

Figure 22 continued Acetylcholine neurotranmission genes Gene Tip /ub nf Title ChatWYSYYSSSSSSSSS SSSSSSSSSSSSSSSSSSS9848 . 0 4 . 444YYYYYYYYYYYYYYYYYYYYYcholine acetyl - transferase Ache 2 . 1 5 acetylcholinesteraseismini . . Slc18a3 16 . 8 6 Vesicular ACH transporter Slc5a7 26 . 4 * * . * Choline. * * * * . *. * . * . * . . * . * .* . * . * . * . * . * ...... transporter+ ...... * . * .* .* . * * * . * * .* * * * *

Peptide TransmittersTTTTTTTTTT Calca 3 .837 CGRP , alpha Calcb 2177. CGRP , beta

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Figure 34 BACarray Molecular Phenotyping of Cerebellar Cell Types in ATM KO Mice

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Figure 39

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Figure 40

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Sphingosine US 9 ,816 ,096 B2 METHODS AND COMPOSITIONS FOR erence to the same extent as if each individual publication , TRANSLATIONAL PROFILING AND patent, or patent application was specifically and individu MOLECULAR PHENOTYPING ally indicated to be incorporated by reference . CROSS -REFERENCE SUMMARY OF THE INVENTION The present application is a continuation of U . S . patent The invention described herein provides methods, com application Ser. No . 12 /918 ,761 , filed Nov . 15 , 2010 , which positions, and kits useful in elucidating the biological prop was the National Stage of International Application No . erties of distinct cellular populations . The embodiments PCT /US09 /01614 , filed Mar. 12 , 2009 , which claims the 10 described herein comprise a translating ribosome affinity benefit of U . S . Provisional Application Nos . 61 /036 ,049 purification ( TRAP ) methodology, which is a generalizable filed Mar. 12 , 2008 , 61 /036 , 058 filed Mar. 12 , 2008 , 61/ 070 . method useful for the identification of molecular changes in 327 , filed Mar . 21 , 2008 , and 61/ 199 ,108 filed Nov . 12 , 2008 any genetically defined cell type in response to genetic which applications are incorporated herein by reference in alterations, disease , environmental, pharmacological, other their entirety . perturbations . In one aspect , the invention described herein provides a SEQUENCE LISTING method of identifying a co -regulated gene set for a function , comprising determining translational profiles for a plurality In accordance with 37 CFR 1 .52 ( e ) (5 ) , a Sequence Listing 20 of cell types that express a gene associated with that func in the form of a text file ( entitled “ 2011060 -0010 ST25” , 20 tion , comparing the translational profiles to determine what created on Jan . 30 , 2017 , and having a size of 9 ,250 bytes) additional genes are similarly regulated , thereby identifying is hereby incorporated by reference in its entirety . a co -regulated gene set involved in that function specifically . STATEMENT AS TO FEDERALLY SPONSORED In one embodiment, the method is provided to identify a RESEARCH 25 co - regulated gene set involved in myelination , and in a further embodiment the gene associated with myelination is This invention was made with the support of the U . S . Mbp . In one embodiment, the co - regulated gene set com Government under Grant No: AG09464 awarded by the prises at least two genes listed in Table 9 . In a related National Institute on Aging , MH074866 awarded by the embodiment, the gene set comprises at least two genes National Institute of Mental Health , DA10044 and 30 selected from the group consisting of Pip1 , Cnp , Mog , Mal 5F32DA021487 awarded by the National Institute on Drug and Mobp . Abuse , 5ULIRR024143 awarded by the National Institutes In another aspect, the invention described herein provides of Health /National Center for Research Resources, and a method for detecting one or more cell type - enriched genes NS34696 awarded by the National Institute of Neurological not detectable by the whole - tissue microarray technique Disorders and Stroke . The U . S . Government may have 35 comprising assessing a translational profile for a cell type , certain rights to the subject matter provided herein . comparing the translational profile to the results of a whole tissue microarray , and determining the presence of one or BACKGROUND OF THE INVENTION more genes in the translational profile that are not present in the results of a whole - tissue microarray , thereby detecting A paradigm in the development of new diagnostics and 40 one or more cell type -enriched genes that are not detectable therapies for human diseases and disorders is the character by the whole -tissue microarray technique . In particular ization of the gene expression of defined cell types . The embodiments , greater than 20 % , 30 % or 40 % of the genes cellular complexity of many tissues poses a challenge for enriched in that cell type are not detectable by the whole those seeking to characterize gene expression at this level. tissue microarray technique . These methods can be applied The enormous heterogeneity of a tissue such as the nervous 45 to any cell type of interest including neuronal and non system ( thousands of neuronal cell types, with non - neuronal neuronal cell types. In exemplary embodiments , the cell type cells outnumbering neuronal cells by an order of magnitude ) is selected from the group consisting of striatal cell, cer is a barrier to the identification and analysis of gene tran - ebellar cell , cortical cell , hypothalamic cell , hippocampal scripts present in individual cell types . Cellular subtypes are cell , brainstem cell, and a spinal cord cell. highly heterogeneous and often intermixed . Gene expression 50 In another aspect , the invention described herein provides studies on isolated cells have been limited by stresses a method of identifying medium spiny neuron - enriched introduced during cellular isolation procedures, the adapta - mRNAs, the method comprising expressing a ribosomal tions which occur upon the loss of tissue - intrinsic signals protein regulated by a regulatory region specific to a gene that control cellular physiology in vivo , and the technical expressed in medium spiny neurons in an organism , isolat challenges associated with reproducible mRNA purification 55 ing complexes comprising the ribosomal protein associated from fixed tissue . A method to isolate translated mRNAs with mRNAs, identifying the mRNAs in the complexes, and from defined cell types and subtypes without the need for comparing the mRNAs to those identified from a reference cell isolation is needed in order to define previously unde - sample . In one embodiment the regulatory region used for fined cell types , identify molecular targets for diseases and expression is specific to a gene expressed in striatonigral disorders , and provide a way in which to identify co - 60 neurons or striatopallidal neurons. In some embodiments the regulated gene sets for a particular biological function . The medium spiny neuron is associated with a disease or disor invention is described here . der such as but not limited to Parkinson ' s disease , addiction , attention deficit hyperactivity disorder, or Huntington 's dis INCORPORATION BY REFERENCE ease . In an additional embodiment, the method can be used 65 in guiding the selection of candidate targets for the treatment All publications , patents , and patent applications men of the disease or disorder , and for further screening of tioned in this specification are herein incorporated by ref- potential modulators of the candidate targets . US 9 ,816 ,096 B2 In yet another aspect, the invention described herein (Fth1 ) out of non -polysome fractions into polysome frac provides a method of identifying motor neuron -enriched tions was observed , as determined by reverse transcription mRNAs, the method comprising expressing a ribosomal followed by quantitative PCR of total RNA purified from protein regulated by a regulatory region specific to a gene non - polysome and polysome gradient fractions (range of expressed in motor neurons , isolating complexes comprising 5 fold - change in non - polysome fraction : 0 . 16 - 1 .22 ; range of the ribosomal protein associated with mRNAs , identifying fold - change in polysome fraction : 1 . 83 - 2 . 15 ). Panel d of the mRNAs in the complexes , and comparing the mRNAs to FIG . 3 shows immunoprecipitations performed from non those identified from a reference sample. In a specific polysome or polysome gradient fractions and 0 . 2 % of the embodiment, the regulatory region comprises regulatory Input ( IN ) , 0 .02 % of the unbound (UB ) ; 1 % of the bound sequences from a choline acetyltransferase (Chat ) locus . 10 (IP ) samples were loaded onto gels for immunoblot analysis This method can be utilized to identify mRNAS from any with eGFP or Rp17 antibodies ; * indicates the presence of a motor neuron , such as but not limited to brain stem motor weak Rp17 band upon much longer exposure . Rp17 was not neurons, spinal cord motor neurons, upper motor neurons present in the non - polysome fraction , presumably because , and others . unlike the overexpressed eGFP -Rp110a , it was all incorpo In another aspect, the invention described herein provides 15 rated into polysomes. As expected from the lack of Rp17 a method for assessing the translational profile of a cerebel - ( and thus assembled ribosomes ) in the non -polysome frac lar cell, comprising expressing a ribosomal protein regulated tion , immunoprecipitations from non -polysome fractions by a regulatory region specific to a cerebellar gene in the did not pull down any RNA above background , indicating cell , isolating at least one complex comprising the ribosomal that the immunoprecipitation was specific to translated mes protein associated with a mRNA from the cell , and identi - 20 sages. Panel e of FIG . 3 shows a direct eGFP immunopre fying the mRNA from the complex whereby generating a cipitation of post- mitochondrial supernatants (whole cell translational profile . In one embodiment, the cerebellar cell lysates , unfractionated ) performed to determine if the trans is associated with a disease or disorder, such as ataxia . lating ribosome affinity purification ( TRAP ) methodology The present invention also provides for kits comprising a could faithfully reflect the changes observed in Panel c of recombinant vector engineered to express a nucleic acid 25 FIG . 3 . 0 . 5 % of input (IN ) , 0 . 5 % of the unbound fraction sequence encoding a ribosomal protein and a detectable tag (UB ) , and 1 . 0 % of the bound (IP ) samples were loaded onto operably linked to an endogenous regulatory region , useful gels for immunoblot analysis with eGFP ( top pane of Panel for practicing the methods described herein . e of FIG . 3 ), Rp110a (middle pane of Panel e of FIG . 3 ), or Rp17 (bottom pane of Panel e of FIG . 3 ) antibodies. * BRIEF DESCRIPTION OF THE DRAWINGS 30 indicates the presence of a light eGFP -L10a band upon longer exposure . No endogenous Rp110a or Rp17 was recov The novel features of the invention are set forth with ered in the bound ( IP ) fraction of mock samples , while particularity in the appended claims. A better understanding endogenous Rp110a and Rp17 were both recovered in the of the features and advantages of the present invention will bound (IP ) fraction of untreated and iron - treated samples . be obtained by reference to the following detailed descrip - 35 Panel fof FIG . 3 shows the iron - induced fold -change in Fth1 tion that sets forth illustrative embodiments , in which the mRNA levels relative to Actb mRNA levels in samples principles of the invention are utilized , and the accompany - immunoprecipitated ( IP ) from either polysomes or from ing drawings of which : whole cell lysates ( range of fold - change in polysome IP FIG . 1 illustrates a CNS disease -related BACarray col - samples: 2 .01 - 2 .43 ; range of fold - change in direct IP lection . 40 samples : 1 . 80 - 1 . 93 ) was similar to the change observed in FIG . 2 presents the translating ribosome affinity purifica - the polysome gradient fraction before immunoprecipitation tion ( TRAP ) methodology. (a ) Schematic of affinity purifi ( c ). cation of GFP tagged polysomes (originating from the FIG . 4 illustrates the in vivo BACarray strategy target cell population ) using anti GFP antibody coated FIG . 5 illustrates BAC vector engineering . beads. (b ) Transmission electron micrographs of anti GFP 45 FIG . 6 illustrates BACarray mouse generation . coated magnetic beads after incubation with extracts taken FIG . 7 illustrates examples of drivers used to produce from HEK293T cells transfected with an empty vector ( left BACarray mice with specific expression in the cerebral panel ) or the eGFP L10a construct ( right panel ) ; images cortex . acquired at 50 ,000x magnification , inserts enlarged by a FIG . 8 illustrates examples of drivers used to produce factor of 2 .3x . 50 BACarray mice with specific expression in the hypothala FIG . 3 illustrates immunoprecipitation of translated mus. mRNAs from transfected cells . FIG . 3 is comprised of FIG . 9 Panels 1 - 25 illustrates that BAC transgenesis Panels a , b , c , d , e , and f. Panel a of FIG . 3 shows that targets eGFP -L10a to specific CNS cell populations . DAB induction of the iron - storage protein Ferritin was seen after immunohistochemistry with anti - eGFP antibody on each 36 hours of iron treatment. Panel b of FIG . 3 shows 55 mouse line reveals a unique and specific pattern of expres post -mitochondrial supernatants of untreated or iron - treated sion for the eGFP -L10a transgene . Panels ( 10x ) show the cells loaded onto linear sucrose gradients ( 20 - 50 % w / w ) . morphology and localization of cell types expressing the After velocity sedimentation , fractions (direction of sedi- transgene, while inset shows location of panel, for cerebellar mentation noted by arrow ) were collected while UV absor - (1 - 9 ) , spinal cord ( 10 ) , striatal/ basal forebrain ( 11 - 14 ) brain bance (254 nm ) was being measured . A representative trace 60 stem ( 15 ) , and cortical ( 16 - 25 ) cell types . Dashed lines is shown , as profiles from non - treated and iron - treated (panels 16 - 25 ) indicate corpus callosum . A key for all cell lysates looked nearly identical. Non - polysome and poly - types is in FIG . 11A . some gradient fractions were generated as indicated . To FIG . 10 illustrates that GENSTAT EGFP and BACarray avoid Ferritin mRNAs associated with mRNPs , only heavier cortical pyramidal cell lines have same laminar distribution . polysomes (with greater than 4 ribosomes ) were included in 65 A ) Graph (mean + / - SE ) of the distance of eGFP + cell somas the polysome fraction . Panel c of FIG . 3 shows that after iron from the pial surface for the BACarray and GENSAT eGFP treatment, as expected , a shift of Ferritin heavy chain mRNA lines for each pyramidal cell BAC driver . The depth of the US 9 ,816 ,096 B2 cells was consistent between both lines for each driver . B ) C , and D . Panel A of FIG . 17 shows that replicates for the The percentage of cells in 100 micron bins is shown as a same cell type gave nearly identical genome wide transla histogram of the distribution of cell depths for each line in tional profiles, confirming the results of Heiman et al, and A . The BACarray line and eGFP line for each driver had extending this finding to many other cell types . The average overlapping distributions of cell depths . 5 Pearson ' s correlation between replicates for a given cell FIG . 11 illustrates a summary of cell types studied and population from independently isolated samples was above characterization of lines. A ) Table of all cell populations 0 . 98 across all cell types . Panel B of FIG . 17 shows scatter studied with BACarray, corresponding to FIG . 9 . B ) Detec - plots for three representative cell types of the cerebellum . tion of eGFP -L10a under the control of a Pcp2 BAC in Panel C of FIG . 17 shows Venn diagrams constructed from Calbindin positive Purkinje cells . C ) Detection of GFP - 10 the top 1000 most enriched probesets for each cell type . L10a under the control of a Neurod1 BAC in granule cells Panel D of FIG . 17 presents an examination of results with NeuN positive nuclei. D ) Detection of eGFPL10a obtained from independent BACarray founder lines pre under the control of a Lypd6 BAC in Parvalbumin positive pared with the same engineered BAC to determine whether outer stellate and deep stellate (basket ) neurons of the the position of integration of the BACarray construct would molecular layer, but not in Parvalbumin positive Purkinje 15 influence the data . cell fibers ( arrow ). E ) Detection of eGFP - L10a under the FIG . 18 illustrates that BACarrays can identify known control of a Grm2 BAC in Grm 2 / 3 positive interneurons markers , and discover new ones for a variety of cell types . (Golgi cells ) of the granule cell layer, but not in Grm2/ 3 FIG . 18 is comprised of Panels A , B , C , and D . Panel A of positive glomeruli (arrow ). F ) eGFP -L10a detected under FIG . 18 shows a scatter plot of IP vs. UB for spinal cord the control of a Grp BAC in unipolar brush cells with Grml 20 motor neurons . Probesets for known markers of motor positive brushes ( arrow ) . G ) eGFP -L10a detected under the neurons with measurable signal on the array are clearly control of a Sept4 BAC in s100 positive Bergman glia . enriched in the IP sample , whereas probesets for glial FIG . 12 illustrates that higher transgene expression yields cell -specific RNAs, that should not be present in these cells , better signal to noise . A ) DAB immunohistochemistry for are enriched in the UB sample . To establish the generality of eGFP -L10a reveals differential intensity of transgene 25 this finding , the enrichment in the IP or UB sample was expression in two BACarray lines targeting the same cell quantified by calculating an average ratio of IP /UB for population . B ) Quality of BACarray data , as assessed by positive and negative controls for each cell type where at average IP /UB fold change for positive and negative con - least three positive controls could be found in the literature . trols , is better for the line with higher transgene expression . Panel B of FIG . 18 shows that all IPs showed a clear FIG . 13 illustrates protein and mRNA purification from 30 enrichment for appropriate known markers ( plotted in log BACarray lines. ( a ) Purification of eGFP tagged L10a and co base 2 ) . Even for cell types with only one known marker purification of untagged ribosomal protein L7 from D1 (Pnoc positive interneurons, and Grp expressing unipolar BACarray animals but not wild type littermates (D1 , brush cells ) , probesets for these genes were consistently and samples from D1 BACarray mice ; WT, samples from wild highly enriched in the IP . In the IPs with the lowest relative type littermates ; In , 1 % Input; UB , 1 % Unbound ; IP , 6 . 5 % 35 yield of RNA , such as those for mature oligodendrocytes Immunoaffinity purified sample ) . eGFP L10a signal is only (Panel B of FIG . 18 ) , and Cort expressing interneurons , present in the D1 IP lane because the IP samples were more background was proportionally higher, and enrichment was concentrated relative to In and UB . ( b ) Purification of 18S less robust. Panel C of FIG . 18 shows that in the case of and 28S rRNA from D1 BACarray transgenic animals ( top cerebellar Golgi cells , there is a great deal of overlap panel) but not wild type littermates (bottom panel ) as 40 between eGFP -L10a expression in the BACarray line and detected by Bioanalyzer PicoChips (Agilent Technologies ) . expression of the genes chosen for this analysis . Panel D of 28S rRNA runs at ~ 47 sec, 18S rRNA runs at ~ 43 sec , and FIG . 18 shows results of quantitative real time PCR (QRT the marker peak runs at ~ 23 sec . PCR ) measuring the enrichment of a variety of mRNAs FIG . 14 illustrates electron microscopy of immunopre isolated from the Chat (motor neuron ) and Pcp2 (Purkinje cipitated ribosome complexes . 45 cell ) BACarray transgenic lines in order to further validate FIG . 15 illustrates that microarray signal intensity does the BACarray datasets . For all of the control genes tested , not increase with transcript length . A -DD : To assess whether this methodology confirmed the BACarray results . For longer mRNA ' s were more efficiently immunoprecipitated genes not previously known to be expressed in a specific cell than shorter transcripts , transcript length ( Y - axis , log base type , results from qRTPCR demonstrated that seven out of 2 ) , was plotted versus GCRMA normalized signal intensity 50 the eight mRNAs assayed were in fact cell type enriched ( X -axis , log base 2 ) for all 30 samples . None of the immu - (Panel D of FIG . 18 ) . Moreover , despite a negative ISH noprecipitated nor the unbound (whole tissue RNA ) samples result , qRT- PCR validated the expression of Ceacam10 in show positive correlations between length and signal. the cerebellum and its enrichment in Golgi cells (Panel D of FIG . 16 illustrates that BACarray can consistently purify FIG . 18 ) . In some cases, therefore , the translating ribosome RNA efficiently from rare and common cell types . A ) 55 affinity purification ( TRAP ) methodology appears to be Western blot for eGFP from $ 20 fraction , flow through more sensitive than ISH . (UB ) , and immunoprecipitate from rare (unipolar brush FIG . 19 illustrates that BACarray clusters cells by type cells ) , common (Purkinje cells ) and extremely common and provides greater sensitivity than whole tissue arrays . ( granule cells ) cell types of cerebellum . Note that even when FIG . 19 is comprised of Panels A , B , and C . Panel A of FIG . protein is undetectable , there is sufficient good quality RNA 60 19 shows hierarchical clustering of cell types and tissue for microarray amplification . B ) Yield of RNA is consistent samples . Panel B of FIG . 19 shows relative number of across replicates within each line. C ) Total RNA is of good probesets detected by Bacarray. Panel C of FIG . 19 shows quality for all three cell types ,with intact 18s and 28s bands, enrichment of genes undetected by whole tissue array . as well as mRNA which can be amplified and fragmented FIG . 20 illustrates cell type diversity is driven by proteins following standard protocols . 65 on the cell surface , such as receptors and ion channels . FIG . FIG . 17 illustrates that BACarray data are reproducible 20 is comprised of Panels A , B , and C . Panel A of FIG . 20 and cell - type specific . FIG . 17 is comprised of Panels A , B , shows average Shannon entropy of BACarray samples. US 9 ,816 ,096 B2 Panel B of FIG . 20 shows examples of probeset with low and longest one was chosen . RefSeq lengths were plotted against high information . Panel C of FIG . 20 shows the ten percent D1 ( a ) or D2 (b ) BACarray IP normalized expression values . of the probesets with highest entropy and those with the There was no correlation observed between transcript length lowest entropy classified with Gene Ontologies and then and IP values . Striatal expression values for all Affymetrix searched for functional categories that were over- repre - 5 Genechip probe sets were obtained by total RNA arrays sented . from wild type striatal tissue (data not shown ) . These values FIG . 21 illustrates that a comparative analysis reveals were plotted against ( c) D1 BACarray or (d ) 02 BACarray unique translational profiles for each cell type. An iterative comparison was performed : one -by -one , each sample was IP normalized values . As expected , higher expression in total compared to each other sample in the dataset, and for each 10 striatum (no IP , wild type mice ) correlates with higher D1 or population , probesets were sorted by their average ranking D2 BACarray IP values. The few genes that show modest across these comparisons. FIG . 21 is comprised of Panels A expression in total striatum but have low IP values include and B . Panel A of FIG . 21 shows data combined and known non neuronal genes. clustered by expression the top one hundred ranked probe FIG . 28 illustrates gene expression analysis of BACarray sets for each population in a heatmap . This heat map readily 15 purifiedpun mRNA . Normalized expression values from illustrates the extent to which distinct cell types are charac Affymetrix Mouse Genome 430 2 . 0 arrays are plotted for D1 terized by specific cohorts of genes. Panel B of FIG . 21 and D2 BACarray samples . Middle diagonal line represents shows the top twenty five most specific probesets in each equal expression , and lines to each side represent 1. 5 fold cell type include probesets for both well -known cell -specific enrichment in either cell population . markers and novel, previously uncharacterized genes. 20 FIG . 29 illustrates expression analysis of medium spiny FIG . 22 illustrates the transcriptional sketch of a spinal neuron (MSN ) enriched genes. Expression analysis in sag motor neuron . ittal sections of genes which were amongst the top 100 (a ) FIG . 23 illustrates expression of eGFP -L10a in the Chat or 1 ,000 1 , 100 ( b ) genes identified in the study as MSN and Purkinje cell BACarray lines . ( A ) Immunohistochem - enriched , with the rank order of each gene noted below the istry to eGFP in adult sagittal sections from the Chat 25 gene name. Non redundant gene ranking was calculated BACarray line DW167. ( B ) Indirect immunofluorescent using the highest ranked probeset corresponding to each characterization of Chat BACarray line DW167 brain stem gene with redundant probesets eliminated . Left panel, in situ facial motor nucleus : eGFP staining ( left panel ); Chat stain - hybridization images taken from the Allen Brain Atlas ing (middle panel ) and merge ( right panel , with 20 um scale (Allen Brain Atlas , Internet] . Seattle (Wash ): Allen Institute bar ) . ( C ) Immunohistochemistry to eGFP in adult sagittal 30 for Brain Science . © 2006 . Available from the hypertext sections from the Pcp2 BACarray line DR166 . ( D ) Indirect transfer protocol on the world wide web , brain -map .org . ) immunofluorescent characterization of Pcp2 BACarray line ( 3 ) ; right panel , in situ hybridization images taken from the DR 166 Purkinje cell neurons: eGFP staining ( left panel) ; Brain Gene Expression Map (BGEM ) database found using Calbindin -D28K staining (middle panel ) and merge ( right the hypertext transfer protocol and the world wide web at panel, with 20 um scale bar ) . 35 stjudebgem . org ) ( 4 ) . Allen Brain Atlas images all corre FIG . 24 illustrates that BACarray profiles recapitulate spond to adult brain ; BGEM images all correspond to adult known cell- specific markers and reveal new ones for four brain except for the following , for which the oldest available distinct cell types . Scatterplots of D1, D2, Chat, and Pcp2 data were postnatal day 7 (P7 ) : Drd2, Ppplrlb , D1x6 , Gdnf, BACarray data compared to a reference mRNA sample Bc111b , Foxgl, Limd2 , Femlb , Dyn111 , Atbf1, Foxo3a , reveal hundreds of genes enriched in each cell type ( A - D ) . 40 Dnalc4 , Mtmr7 , Dnmt3a . Lines on either side of the diagonal mark 2 - fold enrichment. FIG . 30 illustrates that functional Gpr6 receptors are Axes are labeled for expression in powers of 10 . Venn found in BAC D2 striatopallidal neurons but not BAC D1 diagrams of the top 1 , 000 enriched probesets ( Tables 17 - 20 ) striatonigral medium spiny neurons. ( a ) Projection of an ( with expression value cut- off > 100 ) for each cell type reveal eGFP labeled medium spiny neuron from a BAC D2mouse . that each cell type has a unique pattern of enriched genes 45 The cell was patched with a pipette containing Alexa 594 ( 50 ( E - H ) . UM ) for visualization and Fluo 4 (200 M ) for measuring FIG . 25 illustrates expression of eGFP L10a in D1 and D2 changes in intracellular Ca2 + ( right) . Cells were voltage BACarray lines. ( a ) Immunohistochemistry to eGFP in adult clamped at - 70 mV. A puffer pipette containing sphingosine sagittal sections from the D2 BACarray line CP101. ( b ) 1 phosphate ( S1P , 10 UM ) was positioned near a dendrite , 60 Characterization of D2 BACarray line CP101 striatal MSN 50 80 um from the soma ( left/ cartoon ) . ( b ) High magnification cells : direct eGFP fluorescence (left panel with high mag - images of a dendritic segment ( control, left panel) show an nification image insert ) ; enkephalin immunohistochemical increase in Ca2 + associated with SIP application (SIP puff , staining (middle panel) ; merge ( right panel, with 20 um center panel) that reversed with washing (wash , right panel ) . scale bar ). (c ) Immunohistochemistry to eGFP in adult The change in Ca2 + was determined by calculating the sagittal sections from the D1 BACarray line CP73 . ( d ) 55 percent change in fluorescence of Fluo 4 relative to that of Characterization of D1 BACarray line CP73 striatal MSN Alexa 594 (AG / R ) . ( c ) Time course showing the SIP induced cells : direct eGFP fluorescence ( left panel ) ; enkephalin increase in intracellular Ca2 + in the ROI from b (orange immunohistochemical staining (middle panel) ; merge ( right trace ); similar recordings from BAC D1 medium spiny panel) . neurons (black trace ) or thapsigargin loaded BAC D2 FIG . 26 illustrates the polysome profile from D2 BAC - 60 medium spiny neurons did not reveal any changes in den array mouse striatal extracts . dritic Ca2 + levels with S1P application . ( d ) Box plot sum FIG . 27 illustrates an analysis of striatal MSN IP values marizing the S1P effects . Percent increase in fluorescence relative to message abundance and length . Lengths of tran (AG / R ) in BAC D2 medium spiny neurons (median = 146 % , scripts were based on all available mouse curated RefSeq range 44 to 294 % , n = 6 ) ; BAC D1 medium spiny neurons RNA sequences (available using the file transfer protocol, 65 (median = 17 % , range 13 to 22 % , n = 4 ); and thapsigargin ncbi. nih . gov / genomes / M _ musculus /RNA ) . Where multiple loaded BAC D2 medium spiny neurons (median = 4 % , range transcript variants for a single gene were available , the 9 to 10 % , n = 4 ). US 9 ,816 ,096 B2 10 FIG . 31 illustrates that cocaine treatment increases the change between groups in the (c ) mean unitary conductance frequency of small amplitude GABAergic mIPSCs in BAC ( referred to as g in the y -axis ) of GABAA receptors of D1 striatonigral neurons. (a ) Representative spontaneous striatopallidal neurons (t test , p > 0 .05 , Saline g = 30 .7 + 1. 5 , mIPSCs traces from BAC D1 striatonigral neurons ( express- n = 12 ; Cocaine g = 30 . 3 1 . 2 , n = 16 ) or in the ( d ) mean number ing soluble eGFP under the D1 promoter) taken from mice 5 of channels per synapse in striatopallidal neurons taken from treated for 15 days with saline or ( b ) cocaine (20 mg/ kg / cocaine treated BAC D2 mice (t test , p > 0 .05 , Saline day ) . ( c ) Bar graph summary of mean mIPSC frequency showing a increase in BAC D1 striatonigral neuron mIPSCs N = 33 . 8 + 2 . 7 , n = 12 ; Cocaine N = 33 . 0 - 1 . 6 , n = 16 ) . frequency following cocaine treatment (Mann Whitney FIG . 34 illustrates BACarray molecular phenotyping of Rank Sum Test, p < 0 .05 , saline median = 0 .82 Hz, n = 22 ; 10 cerebellar cell types in ATM KO mice . cocaine median = 1 . 03 Hz, n = 26 ) . ( d ) Bar graph summary FIG . 35 illustrates the characterization of Pcp2 and Sept4 showing that the number of small amplitude mIPSCs (< 75 BACarray transgenic mouse lines. ( A ) Expression of the PA ) in equal length records ( 7 min ) increased in BAC D1 eGFP L10a fusion in Purkinje cells in the cerebellum of the striatonigral neurons following cocaine treatment (t test , Pcp2 BACarray line. Immunohistochemical stain of Pcp2 p < 0 .05 . saline = 79. 4 + 18 . 2 . n = 22 : cocaine= 104 . 2 + 6 . 3 . n = 26 ) . 15 BACarray brain section using anti eGFP antiserum (top ( e ) Representative variance mean current plots from saline panel) . Double immuno fluorescence analysis (bottom pan treated and cocaine treated BAC D1 neurons suggesting that els ) of calbindin (Calbl ) and eGFP L10a expression in Pcp2 the cocaine induced small amplitude events arise from BA?array mice showing co labeling of Purkinje cells synapses that have fewer GABAA receptors ( N ) per synapse (merge ) . ( B ) Expression of the eGFP L10a fusion in Berg but receptors with an unchanged unitary receptor conduc - 20 mann glia in the cerebellum of the Sept4 BACarray line . tance ( g ) (saline N = 33 , g = 31 pS ; cocaine N = 27 , g = 30 PS ; Immunohistochemical stain of Sept4 BACarray brain sec see FIG . S6c d for means) . ( f ) Representative spontaneous tion using anti eGFP antiserum ( top panel) . Double immuno mIPSCs traces from BAC D2 striatopallidal neurons fol fluorescence analysis of glutamine synthetase (Gins ) and lowing saline treatment for 15 days and ( g ) following eGFP L10a expression in Sept4 BACarray mice showing co cocaine treatment for 15 days . ( h ) Bar graph summary of 25 labeling of Bergmann glial cells (merge ) . mean mlPSC frequency in saline and cocaine treated neu FIG . 36 illustrates a scatter Plot analysis of BACarray rons, showing no effect of treatment condition ( t test, data for Septin4 and Pcp2 lines . Immunoprecipitated (IP ) p > 0 .05 , saline = 0 .72 : 0 .05 Hz, n = 12 ; cocaine = 0 .78 = 0 .07 Hz , mRNA from Septin4 ( A , C ) and Pcp2 ( B , D ) BAC trans n = 16 ) . ( i ) Bar graph summary showing that the number of genic lines was directly compared in scatter plot analysis to small amplitude mIPSCs ( < 75 PA ) in equal length records ( 7 30 min ) was not altered by treatment condition in BAC D2 mRNA samples from total cerebellum . The analysis clearly neurons ( t test, p > 0 .05 , saline = 68 . 109. 7 , n = 12 ; demonstrates that thousands of genes are enriched in the cocaine = 71. 7 + 7 . 5 , n = 16 ) . ( 1 ) Representative variance mean immunoprecipitated samples relative to total cerebellum . current plots showing that cocaine treatment did not change Scatter plots ( C ) and (D ) show the same experiments with in the number of receptors per synapse or the unitary 35 known positive control genes for Bergmann glial cells and receptor conductance in BAC D2 neurons ( saline N = 29 , Purkinje neurons. Bergmann glial positive controls are g = 33 pS ; cocaine N = 31 , g = 29 pS ; ) . clearly enriched in the Septin4 IP samples ( C ) whereas FIG . 32 illustrates that cocaine treatment decreases the Purkinje cell positive control genes are enriched in Pcp2 IP numberer of GABAA channels per synapsesynapse in BAC D1 samples ( D ) . striatonigral neurons . Box plot summaries showing mean 40 FIG . 37 illustrates cell type specific differential regulation mIPSCs kinetics and non stationary noise analysis measures of gene expression in Atm - / - cerebellum . ( A - C ) Atm - / - IP of individual striatonigral neurons taken from saline treated data were compared to Atm + / + IP data in scatter plot analy control and 15 day treated cocaine BAC D1 mice . ( a ) ses for the Septin4 ( A ), the Pcp2 (B ) and the total cerebellum Neither mean 10 90 % rise times ( t test , p > 0 .05 , ( C ) experiments . Gene lists of both upregulated and down Saline = 0 .64 + 0 . 008 , n = 22 ; Cocaine = 0 .65 + 0 .006 , n = 26 ) nor 45 regulated genes were generated by filtering on expression ( b ) mean decay times ( ttest, p > 0 .05 , Saline = 17 . 3 + 0 . 5 , n = 22 ; values greater than 25 , on fold change values greater than Cocaine = 17 . 0 + 0 . 5 , n = 26 ) were different between groups. 1 . 5 , and performing one way ANOVA , p = 0 . 05 . Scatter plots Non stationary noise analysis of mIPSCs demonstrated no display the distribution of regulated genes in each experi change between groups in the ( c ) mean unitary conductance ment. Colors refer to the Venn Diagram analysis ( D ) . Genes ( g ) of GABAA receptors of striatonigral neurons ( t test , 50 shared by Bergmann glia and Purkinje cells are colored p > 0 .05 , Saline g - = 31. 1 - 1 . 6 , n = 22 ; Cocaine g = 30 . 8 - 1 . 2 , yellow , those shared by Purkinje cells and total cerebellum n = 26 ), and a decrease in the ( d ) mean number of channels are turquoise, and those shared by Bergmann glia and total per synapse in striatonigral neurons taken from cocaine cerebellum are colored magenta . Genes identified in all three treated BAC D1 mice ( t test, p < 0 . 05 , Saline N = 33 . 1 + 1. 4 , conditions are colored white . (D ) Venn Diagram analysis of n = 22 ; Cocaine N = 29 . 3 - 1 . 0 , n = 26 ) . 55 all regulated genes . In total cerebellum , 40 /69 genes are FIG . 33 illustrates that cocaine treatment does not alter found differentially regulated exclusively in total cerebel GABAergic mIPSCs kinetics or GABAA receptor number lum , in the Septin4 experiment, 94 / 116 genes are specifically per synapse or unitary receptor conductance in BAC D2 regulated in the Septin4 data , and in the Pcp2 experiment, striatopallidal neurons . Box plot summaries showing mean 62 / 89 regulated genes which are specific to the Pcp2 data mIPSCs kinetics and non stationary noise analysis measures 60 have been identified . of individual striatopallidal neurons taken from saline FIG . 38 illustrates ventral tegmental area and substantia treated control and 15 day treated cocaine BAC D2 . ( a ) nigra area specific markers , as revealed by BACarrays . Neither mean 10 90 % rise times ( t test , p > 0 .05 , FIG . 39 illustrates dopaminergic cell loss in the rodent Saline = 0 .65 + 0 .007 , n = 12 ; Cocaine = 0 .6410 .010 , n = 16 ) nor substantia nigra with the use of 6 -OHDA . ( b ) mean decay times ( t test, p > 0 . 05 , Saline = 18 . 0 - 1 . 0 , n = 12 ; 65 FIG . 40 illustrates a therapeutic opportunity for Parkin Cocaine = 18 . 0 + 0 . 5 , n = 16 ) were different between groups. son ' s disease , by developing new strategies for stimulation Non stationary noise analysis of mIPSCs demonstrated no o f Sub - Thalamic Nucleus, by using antagonists of Gpr6 . US 9 ,816 ,096 B2 12 DETAILED DESCRIPTION OF THE tinguishing molecular characteristics of closely related or INVENTION critical cell types. Methods also demonstrate that methods described herein can be employed to analyze physiological Translational Profiling and Molecular Phenotyping adaptations of specific cell types in vitro , in vivo , ex vivo , The present disclosure provides for methods and compo - 5 or in situ . sitions useful in translational profiling and molecular phe - Molecular Tagging of Ribosomes notyping of heterogeneous tissues and cell types . In one Embodiments of this invention provide methods for iso embodiment the methods can be used to define previously lating cell type - or cell subtype- specific polysomal, mRNA . undefined cell types , identify molecular targets for diseases The methods include using molecularly tagged ribosomal and disorders , and provide a way in which to identify 10 proteins resulting in functional tagged ribosomes or ribo co - regulated gene sets for particular biological and / or medi- somal complexes , able to support translation , assemble into cally relevant functions . polysomal complexes. Ribosomes can be molecularly Translational profiling is the profiling, identification , or tagged and expressed in one or more cell types or cell isolation of translated mRNAs. In some embodiments such subtypes of interest .Molecularly tagged ribosomal proteins profiling is a measure of the nascent proteome. In other 15 for the purposes of this disclosure will interchangeably be embodiments , this profiling allows for the identification of referred to as a ' fusion proteins ' or ' tagged proteins ' or mRNAs being actively translated , or otherwise associated molecularly tagged fusion proteins ' or 'molecularly tagged with the cellular translational machinery. Molecular pheno - ribosomal proteins '. In various embodiments these fusion typing is the molecular and / or gene expression description proteins contain all or a portion of a ribosomal proteins. In of organs , tissues , and cell types . 20 certain embodiments ribosome tagging causes no disruption The present disclosure provides for methods and compo - of native function or distribution of the tagged protein , thus sitions to practice translating ribosome affinity purification resulting in a functional ribosome, functional ribosomal ( TRAP ) profiling methodology . In one embodiment these complex , or functional polysome. That is , although molecu profiling methods are be utilized to further distinguish larly tagged , the portion that has the biological activity of the morphologically , anatomically , developmentally , or other - 25 native ribosomal protein is retained and can function in an wise indistinguishable, cells into cellular subtypes , further intact ribosome to carry out translation or binding of mRNA . defining cell populations and sub -populations . In some In some embodiments , the molecularly tagged ribosomal is cases , these otherwise indistinguishable cells are intermixed . expressed in a organ , tissue , cell type , or cell subtype of In other cases , these cells are spatially separated . In some interest, by introducing into cells , culture , slices , or into an cases , these cells are cells of the central or peripheral 30 entire organism , a nucleic acid encoding the molecularly nervous system , for example neurons or glia , only distin - tagged ribosomal protein under the control of regulatory guishable by their translational profiles and molecular phe - elements or transcriptional units that direct expression in the notypes . In other cases these are cells outside the nervous cell type or cell subtype of choice . In some embodiments the system . In another embodiment these profiling methods are ribosomal protein to be tagged can be from the same or be utilized to identify translated mRNAs in single cell types 35 different species as the cell that expresses the molecularly with a sensitivity that is may not be achievable by techniques tagged protein . such as whole - tissue microarray . In yet other embodiments , In certain embodiments , the ribosome or ribosomal pro these profiling methods are used to identify co - regulated tein is molecularly tagged by engineering the ribosomal gene sets for particular biological functions in cells that are protein to fuse to or bind a small molecule , a protein , or spatially separated or intermixed , morphologically distinct 40 peptide that is not bound by the unengineered ribosome or or indistinct , in different developmental stages , associated the ribosomal protein . In certain embodiments the nucleic with different tissues or regions of a tissue, or from different acid encoding the ribosomal protein fused to the tag can be individuals with the same disease , disorder or state . generated by routine genetic engineering methods in which The methods provided herein allow for isolation of a nucleotide sequence encoding the amino acid sequence for mRNAs associated with ribosomes or polysomes (clusters of 45 the tag sequence is engineered in frame with the nucleotide ribosomes ) from specific cell types , allowing for cell trans - sequence encoding a ribosomal protein . This can be accom lational profiling and molecular phenotyping of the cell plished by any method known in the art , for example , via types and subtypes . In some cases, the cells are genetically oligonucleotide- mediated site - directed mutagenesis or poly targeted . merase chain reaction ( PCR ) and other routine protocols of The methods described herein in allow for identifying 50 molecular biology (see , e . g . , Sambrook et al . , 2001 , Molecu translated mRNAs in any cell subtype of interest . The lar Cloning , A Laboratory Manual, Third Edition , Cold methodology involves expression of a tagged functional Spring Harbor Laboratory Press , N . Y . ; and Ausubel et al. , ribosomal protein , which enables tagging of polysomes for 1989 , Current Protocols in Molecular Biology, Green Pub purification ofmRNA , in specific cell populations . In some lishing Associates and Wiley Interscience , N . Y . , both of embodiments , the purification of polysomes is by affinity or 55 which are hereby incorporated by reference in their entire immunoaffinity purification . In some embodiments , the cell ties ) . subtypes are genetically targeted . In some embodiments the In exemplary embodiments the ribosomal protein is L10a tagged ribosomes are expressed in transgenic animals . and the tag is eGFP . The translating ribosome affinity purification methodol - 1 ) Ribosomal Proteins ogy ( TRAP ) for translational profiling and molecular phe - 60 Nucleic acids encoding the tagged proteins can be pro notyping involves expression of a tagged ribosomal protein , duced using genetic engineering methods and cloning and which enables tagging of polysomes for affinity -based , expression vectors that are well known in the art. In some sometimes immunoaffinity -based , purification of mRNA, in embodiments , a naturally occurring or synthetically pro specific cell populations. In some cases this is achieved with duced nucleic acid or gene can be engineered to function as the use of animals or transgenic animals , allowing transla - 65 a ribosomal protein and support translation or bind (directly tion profiling and molecular phenotyping from whole ani- or indirectly ) or associate with polysomal, translating ribo mals . Methods provided herein allow for defining the dis - somal mRNA . Nucleic acids encoding the ribosomal protein US 9 ,816 ,096 B2 13 14 to bemolecularly tagged may be obtained using any method or interfere with function of the tagged protein . The tag may known in the art . Nucleic acids may be obtained , for b e an intact naturally occurring or synthetically produced example , by PCR using oligonucleotide primers based upon protein , fragment, analog or derivative thereof of any length the published sequences. Other related ribosomal ( for that permits binding to the corresponding binding reagent/ example from other species ) may be obtained by low , 5 method . In certain embodiments , the tag is about 8 , 10 , 12 , medium or high stringency hybridization of appropriate 15 , 18 or 20 amino acids , is less than 15 , 20 , 25 , 30 , 40 or nucleic acid libraries using the ribosomal in hand as a probe . 50 amino acids , but may be 100 , 150 , 200 , 250 , 300 , 400 , Exemplary ribosomal proteins for use in various embodi 500 , 1000 or more amino acids in length . The tag may be ments of this invention are provided in bound specifically by a reagent that does not otherwise bind Table 1 , but not limited to those listed . In an exemplary 10 any component of: ( 1 ) the cell of interest ; or ( 2 ) a polysomal embodiment, the ribosomal protein is L10a . In some preparation of interest ; or ( 3 ) whatever cellular fraction of embodiments the tagged ribosomal protein is incorporated interest is being contacted by the reagent that binds the tag . into a ribosomal complex and is associated with one or more In certain embodiments, the nucleotide sequence encod mRNAS , but does not bind mRNA directly . ing a tag is preferably inserted in frame such that the tag is In certain embodiments , the ribosomal protein is mouse 15 placed at the N - or C - terminus of the ribosomal protein , ribosomal protein L10a having the amino acid sequence set since these portions of proteins are often accessible to forth in GenBank NM _ 011287 . 1 (SEQ ID NO : 30 ). detection , affinity , immunological, or purification reagents . TABLE 1 Ribosomal Proteins A52 L11 L23a L35a LP2 ( Large P2 ) S11 S24 Ke - 3 L12 L24 L36 LP1 (Large P1 ) S12 S25 L3 L13 L26 L36a S2 S13 S26 L3L ( L3 - like ) L13a L27 L37 S3 S14 S27 L4 L14 L27a L37a S3a S15 S27a L5 L15 L28 L38 S15a S28 L6 L17 L29 L39 S16 S29 L7 L18 L30 L41 S17 S30 L7a L18a L31 L44 S18 S23 L8 L19 L32 LAMR1 S8 S19 RPLP1 LLRep3 vvv$NO9 S20 L9 L21 L32 - 3a (3a ) L10 L22 L34 LPO (Large P0 ) S10 S21 L10a L23 L35 Region containing hypothetical protein FLJ23544

In certain embodiments , a nucleic acid encoding a 35 Alternatively , the tag can be inserted into any portion of the molecularly tagged ribosomal protein is intended for a ribosomal protein such that when the fusion protein is particular expression system , in which the codon frequen - incorporated into an intact ribosome, ribosomal function is cies reflect the tRNA frequencies of the host cell or organism not compromised and the tag is accessible to the reagent/ in which the protein is expressed . Codon optimization can method to be used in the detection , isolation or and /or allow for maximum protein expression by increasing the 40 purification . That is , tagging can still allow for a functional translational efficiency of a gene of interest . In other ribosome. In other embodiments , a ribosomal protein may embodiments the nucleic acid encoding a molecularly be molecularly tagged with a plurality of tags. tagged ribosomal protein may be a synthetic nucleic acid in In some embodiments , the tag is Green Fluorescent Pro which the codons have been optimized for increased expres tein (GFP ) . GFP can be utilized as an optical or immuno sion in the host cell in which it is produced . 45 affinity tag for a variety of applications, all well known in the 2 ) Molecular Tags art. GFP is a protein , composed of 238 amino acids ( 26 . 9 Molecular tags can be any protein (or fragment, portion , kDa ), originally isolated from the jellyfish that fluoresces analog or derivative thereof) that is not present or accessible in the cell of interest ( or the cell fraction from which the green when exposed to blue light ( Prendergast F , Mann K tagged ribosomes are to be isolated , or other cells that will 50 ( 1978 ) . “ Chemical and physical properties of aequorin and be contacted with the reagent that binds the tag ) for which the green fluorescent protein isolated from Aequorea for there exists a reagent ( such as an antibody ) or method ( such skilea ” . Biochemistry 17 ( 17 ) : 3448 -53 ; Tsien R ( 1998 ) . as optical, fluorescence or magnetic sorting ) that recognizes “ The green fluorescent protein ” . Annu Rev Biochem 67 : the tag and that is accessible to solution ( and thereby , the 509 - 44 ) . The GFP from A . victoria has a major excitation tag ) . 55 peak at a wavelengthwavelergui ofo 395 nm and a minor one at 475 nm . Molecular tagging with tags that are traditionally utilized Its emission peak is at 509 nm which is in the lower green as reporter genes is well known in the art ( Current Protocols portion of the visible spectrum . The GFP from the sea pansy in Molecular biology , Section 9 .6 . 1 , “ Uses of Fusion Genes (Renilla reniformis ) has a single major excitation peak at in mammalian Transfection ” (2004 ) ) . Molecular tagging 498 nm . In cell and molecular biology , the GFP gene is with epitopes are also well known in the art (“ epitope 60 frequently used as a reporter of expression (Phillips G tagging " reviewed in Fritze C E , Anderson T R . Epitope (2001 ) . “ Green fluorescent protein — a bright idea for the tagging : general method for tracking recombinant proteins. study of bacterial protein localization ” . FEMS Microbiol Methods Enzymol. 2000 ; 327 : 3 - 16 ; Jarvik J W , Telmer CA . Lett 204 ( 1 ) : 9 - 18 . ) . In modified forms it has been used to Epitope tagging . Annu Rev Genet . 1998 ; 32 : 601 - 18 ) . make biosensors, and many animals have been created that Tags can include those for which methods/ reagents /de - 65 express GFP . The GFP gene can be introduced into organ vices exist that allow facile identification and isolation of the isms and maintained in their genome through breeding , or tagged protein , but do not, minimally , or negligibly inhibit local injection with a viral vector can be used to introduce US 9 ,816 ,096 B2 15 16 the gene . To date , many bacteria , yeast and other fungal Bornhorst et al. , 2000 , Purification of proteins using poly cells , plant, fly , and mammalian cells have been created histidine affinity tags , Methods Enzymol 326 :245 -54 ; Wil using GFP as a marker . son I A , Niman H L , Houghten R A , Cherenson A R , In other embodiments , the tag is a GFP mutant, variant, Connolly M L , Lemer R A . The structure of an antigenic analog, fragment, or derivative thereof. Different mutants of 5 determinant in a protein . Cell . 1984 July ; 37 ( 3 ) : 767 -78 ; GFP have been engineered ( Shaner N , Steinbach P , Tsien R Evan G I , Lewis G K , Ramsay G , Bishop J M . Isolation of ( 2005 ). “ A guide to choosing fluorescent proteins " . Nat monoclonal antibodies specific for human c -myc proto Methods 2 ( 12 ) : 905 - 9 ) . One improvement was a single oncogene product. Mol Cell Biol. 1985 December; 5 ( 12 ) : pointmutation (S65T ) reported in 1995 in Nature by Roger 3610 - 6 ; Wang L F , Yu M , White JR , Eaton B T . BTag : a Tsien (Heim R , Cubitt A , Tsien R ( 1995 ) . “ Improved green " novel six -residue epitope tag for surveillance and purifica fluorescence ” . Nature 373 ( 6516 ) : 663 - 4 . ) ) . The addition of tion of recombinant proteins . Gene . 1996 Feb . 22 ; 169 ( 1 ) : the 37° C . folding efficiency (F64L ) point mutant to the 53 - 8 ; Hopp et al ., U . S . Pat . No. 4 , 703 , 004 , entitled “ Syn scaffold yielded enhanced GFP ( eGFP ) . eGFP has an extinc - thesis of protein with an identification peptide ” issued Oct. tion coefficient (denoted € ), also known as its optical cross 15 27 , 1987 ; Brizzard B L , Chubet R G , Vizard D L . Immu section of 9 .13x10 -21 m²/molecule , also quoted as 55 ,000 noaffinity purification of FLAG epitope- tagged bacterial L /( mol . cm ) (Shelley R . McRae , Christopher L . Brown and alkaline phosphatase using a novel monoclonal antibody and Gillian R . Bushell (May 2005 ) ) . “ Rapid purification of peptide elution . Biotechniques. 1994 April; 16 ( 4 ) : 730 - 5 ; eGFP , EYFP , and ECFP with high yield and purity ” . Protein Knappik A , Pluckthun A . An improved affinity tag based on Expression and Purification 41 ( 1 ) : 121 - 127 . ). Superfolder 20 the FLAG peptide for the detection and purification of GFP , a series of mutations that allow GFP to rapidly fold and recombinantantibody fragments. Biotechniques . 1994 Octo mature even when fused to poorly folding peptides, was ber ; 17 ( 4 ) :754 - 761; Skerra et al. , U . S . Pat. No . 5 ,506 , 121 , reported in 2006 ( Pedelacq J , Cabantous S , Tran T , Terwil - entitled Fusion peptides with binding activity for streptavi liger T , Waldo G (2006 ). “ Engineering and characterization din , issued Apr. 9 , 1996 ; Skerra A , Schmidt T G . Applica of a superfolder green fluorescent protein ” . Nat Biotechnol 25 tions of a peptide ligand for streptavidin : the Strep - tag . 24 ( 1 ) : 79 -88 ) . In an exemplary embodiment the tag is Biomol Eng. 1999 Dec . 31 ; 16 ( 1 - 4 ) :79 -86 ; Skerra A , Sch enhanced GFP ( eGFP ). nudt TG . Use of the Strep - Tag and streptavidin for detection Other mutations have been made, including color and purification of recombinant proteins. Methods Enzymol . mutants ; in particular blue fluorescent protein ( EBFP , 2000 : 326 : 271 - 304 : Shaner N . Steinbach P . Tsien R (2005 ) . EBFP2, Azurite , mKalamal ) , cyan fluorescent protein 30 “ A guide to choosing fluorescent proteins ” . Nat Methods 2 ( ECFP , Cerulean , CyPet ) and yellow fluorescent protein (12 ) : 905 - 9 . doi: 10 . 1038 /nmeth819 . PMID16299475 .; Sha derivatives ( YFP , Citrine, Venus , YPet) . BFP derivatives ner N , Steinbach P , Tsien R (2005 ) . “ A guide to choosing ( except mKalamal ) contain the Y66H substitution . The fluorescent proteins” . Nat Methods 2 ( 12 ): 905 - 9 . mutation in cyan derivatives is the Y66W substitution . The PMID16299475 ; Heim R , Cubitt A , Tsien R ( 1995 ). red -shifted wavelength of the YFP derivatives is accom - 35 “ Improved green fluorescence” . Nature 373 (6516 ) : 663 - 4 . plished by the T203Y mutation ( Tsien R ( 1998 ). “ The green PMID7854443 ; Shelley R . McRae , Christopher L . Brown fluorescent protein ” . Annu Rev Biochem 67 : 509 - 44 ). and Gillian R . Bushell (May 2005 ) . “ Rapid purification of Molecular tags may include both fluorescent, magnetic , eGFP , EYFP . and ECFP with high yield and purity ” . Protein epitope , radioactive or otherwise detectable tags, by way of Expression and Purification 41 ( 1 ) : 121- 127 ; Pedelacq J . example , and not by limitation , those listed in Table 2 . 40 Cabantous S , Tran T . Terwilliger T , Waldo G ( 2006 ) . “ Engi neering and characterization of a superfolder green fluores TABLE 2 cent protein ” . Nat Biotechnol 24 ( 1 ) : 79 - 88 . PMID16369541 ; Miesenböck G , De Angelis D , Rothman J Molecular Tags ( 1998 ) . “ Visualizing secretion and synaptic transmission GFP, eGFP, mGFP, emerald GFP, derivatives and variants (Green 45 with pH - sensitive green fluorescent proteins " . Nature 394 Fluorescent protein ) BFP, eBFP , eBFP2 , Azurite , mKalamal, sapphire GFP, derivatives (6689 ) : 192 -5 . PMID9671304 . and variants ( Blue fluorescent proteins ) Isolation of Ribosomes, Polysomes, mRNA ECFP, Cerulean , CyPet , DFP , TFP ,mTFP , derivatives and variants 1 ) Isolation of Ribosomes ( cyan and teal fluorescent proteins ) Various methods exist to isolate ribosomes / tagged ribo YFP, Citrine, Venus , YPet, Topaz GFP , derivatives and variants (yellow fluorescent proteins ) 50 somes, particularly polysomes ( ribosomal clusters bound to RFP , mCherry , td - tomato , derivatives and variants ( red mRNA )/ tagged polysomes , from cells , cultured cells and fluorescent protein ) tissues ( see , e . g . , Bommer et al. , 1997 , Isolation and char Bgal, derivatives and variants ( Bgalactosdiase ) acterization of eukaryotic polysomes , in Subcellular Frac Luc , derivatives and variants ( Luciferase ) CAT, derivatives and variants ( Chloramphenicol acetyltransferase ) tionation , Graham and Rickwood ( eds . ), IRL Press , Oxford , hGH , derivatives and variants (human growth hormone ) 55 pp . 280 - 285 ; incorporated herein by reference in its Protein A fragments ; entirety ) . Polysomes are interchangeably referred to as Myc epitopes polyribosomes, ribosomal complexes or ribosomal clusters . Btag and polyhistidine tracts Influenza virus hemagglutinin protein , fragments , derivatives, variants In preferred embodiments , the isolated polysomes ( ribo C -myc gene , fragments , derivatives, variants somal- mRNA complexes ) contain functional ribosomes , Bluetongue virus VP7 protein , fragments , derivatives, variants 60 capable of supporting translation , association with mRNA , FLAG peptide , fragments , derivatives , variants and / or association with translation factors . Strep - tag peptide , fragments , derivatives , variants In certain embodiments , the isolation method employed pHlourins , fragment, derivatives, variants has one or more of the following aspects : a . Maintenance of ribosomal subunits on mRNA during References for Table 2 are provided here , and incorpo - 65 isolation : translation arresting compounds, such as rated by reference herein : Evan et al. , Mol . Cell Biol. emetine or cycloheximide can be added to arrest trans 5 ( 12 ): 3610 - 3616 ; Wang et al. , 1996 , Gene 169 ( 1) :53 -58 ; lation , whereby reducing or preventing dissociation of US 9 ,816 ,096 B2 18 mRNA from the ribosome. In preferred embodiments the art. For example , elution of mRNA is accomplished by isolation is achieved without crosslinking and cross addition of EDTA to buffers, which disrupts polysomes and linking reagents ; allows isolation of bound mRNA for analysis (Schutz , et al. b . Inhibition of endogenous RNAase activity : RNAase ( 1977 ) , Nucl. Acids Res . 4 :71 - 84 ; Kraus and Rosenberg inhibitors can be added to buffers to maintain the 5 (1982 ) , Proc . Natl . Acad . Sci . USA 79: 4015 -4019 ) . In addi integrity of the mRNA ; tion , isolated polysomes ( attached or detached from isola c . Isolation of Polysomes : After tissue or cell homogeni- tion matrix ) can be directly inputted into RNA isolation zation , total polysomes are isolated by preparing a procedures using reagents such as Tri - reagent (Sigma ) or post- mitochondrial supernatant in the presence of at Triazol (Sigma ) . In particular embodiments, poly A .sup . + least a high concentration salt buffer , for example about 10 mRNA is preferentially isolated by virtue of its hybridiza 100 - 150 mM KC1; and tion of oligodT cellulose . Methods of mRNA isolation are d . Solubilization of rough ER -bound Polysomes under described , for example , in Sambrook et al. , 2001, Molecular non - denaturing conditions: Detergent can also be Cloning, A Laboratory Manual, Third Edition , Cold Spring added to release membrane- associated polysomes from Harbor Laboratory Press , N . Y .; and Ausubel et al. , 1989 , endoplasmic reticulum membranes ; total polysomes 15 Current Protocols in Molecular Biology , Green Publishing are usually collected by centrifugation through , for Associates and Wiley Interscience , N . Y . , both of which are example , a sucrose cushion . hereby incorporated by reference in their entireties . In other embodiments , variations of the above -described Nucleic Acid Sequences for Cloning , Expression and Regu general method are used to isolate membrane - associated lation of Tagged Ribosomes polysomes from a total pool of polysomes. This allows for 20 1 ) General further enrichment of mRNA encoding secreted or trans - Methods that are well known to those skilled in the art can membrane proteins. Variousmethods may be used to isolate be used to construct vectors containing tagged ribosomal membrane -associated polysomes from cultured cells and protein coding sequences operatively associated with the tissue, e . g . , methods that employ differential centrifugation appropriate transcriptional and translational control signals (Hall C , Lim L . Developmental changes in the composition 25 required for expression . These methods include , for of polyadenylated RNA isolated from free and membrane - example , in vitro recombinant DNA techniques and in vivo bound polyribosomes of the rat forebrain , analyzed by genetic recombination . See, for example , the techniques translation in vitro . Biochem J . 1981 Apr. 15 ; 196 ( 1 ) :327 - described in Sambrook et al. , 2001 , Molecular Cloning, A 36 ) , rate - zonal centrifugation (Rademacher and Steele , Laboratory Manual, Third Edition , Cold Spring Harbor 1986 , Isolation of undegraded free and membrane- bound 30 Laboratory Press , N . Y . ; and Ausubel et al ., 1989 , Current polysomal mRNA from rat brain , J. Neurochem . 47 ( 3 ): 953 - Protocols in Molecular Biology, Green Publishing Associ 957) , isopycnic centrifugation (Mechler , 1987 , Isolation of ates and Wiley Interscience , N . Y . messenger RNA from membrane - bound polysomes , Meth - 2 ) Regulatory Sequences ods Enzymol. 152 : 241- 248 ) , and differential extraction Certain embodiments of the invention provide vectors , ( Bommer et al. , 1997 , Isolation and characterization of 35 cell lines , and lines of organisms that contain nucleic acid eukaryotic polysomes, in Subcellular Fractionation , Graham constructs that comprise the coding sequence for a tagged and Rickwood (eds . ) , IRL Press , Oxford , pp . 280 - 285 ; ribosomal protein under the control of regulatory sequences . incorporated herein by reference in its entirety ) to isolate the According to the methods described herein , the tagged membrane -associated polysomes (Heintz US publication ribosomal proteins are selectively expressed in a particular 20050009028 incorporated in its entirety ) . 40 chosen cell type , cell subtype , molecular pathway , or circuit In other embodiments , affinity methods are used to isolate using regulatory elements naturally occurring or engineered or purify tagged proteins using methods well known in the to recapitulate a desired expression pattern . Such expression art including but not limited to including chromatography, is achieved by driving the expression of the tagged ribo solid phase chromatography precipitation , matrices, co - im somal protein using regulatory sequences or a transcriptional munoprecipitation , etc . 45 unit from a characterizing or endogenously regulated gene In specific embodiments, molecularly tagged functional expressed in the chosen cell type , subtype , pathway , circuit ribosomes are provided , bound to mRNA , that can bind a and /or point in development. In certain embodiments a reagent for the molecular tag . In other embodiments , the particular cell type is one that resides within a mixed molecularly tagged ribosomes are bound to a specific population of cells, comprising a discernable group of cells reagent or affinity reagent that is bound , covalently or 50 sharing a common characteristic . This group may be mor non - covalently , to a solid surface , such as a bead , a resin , or p hologically , anatomically, genetically , or functionally dis a chromatography resin , e . g . , agarose , sepharose , and the cernible . In other embodiments , this group may be morpho like . In other embodiments , other methods are used with or logically , genetically , and developmentally in place of affinity purification . In other embodiments , indistinguishable , but be genetically distinguishable . specific polysomes can be isolated utilizing optical sorting , 55 Because of its selective expression , the population of cells fluorescence -based sorting or magnetic - based sorting meth - may be characterized or recognized based on its positive ods and devices. expression of an endogenously regulated characterizing In certain embodiments , polysomes are not isolated from gene . Some or all of the regulatory sequences /elements may the post- mitochondrial supernatant or even from a cell or be incorporated into nucleic acids ( including transgenes ) to tissue lysate before being subject to affinity purification . 60 regulate the expression of tagged ribosomal protein coding FIG . 2a presents a schematic to illustrate affinity purifi - sequences . Examples of regulatory sequences or elements cation of eGFP - tagged polysomes using anti -GFP antibody - include but are not limited to enhancer sequences, insulator coated beads . sequences, silencer sequences, or a combination of said 2 ) Isolation of mRNA from Ribosomes sequences. In certain embodiments, a gene that is not Once the tagged ribosome has been isolated , the associ - 65 constitutively expressed , ( i . e. , exhibits some spatial or tem ated mRNA complexed with the protein may be isolated poral restriction in its expression pattern ) is used as a source using chemical, mechanical or other methods well known in of a regulatory sequence . In other embodiments , a gene that US 9 ,816 ,096 B2 20 is constitutively expressed is used as a source of a regulatory rotransmitter pathway, a nucleotide receptor- specific path sequence . In other embodiments a regulatory sequence is way, ion channels, transcriptions factors , markers of engineered or modified to achieve desired expression char- undifferentiated or not fully differentiated cells , the sonic acteristics and patterns. hedgehog signaling pathway, calcium binding , or a neuro Regulatory regions can be whole or parts or the regulatory 5 trophic factor receptor ( tables in US2005 /0009028 , herein sequences from the loci of the genes of interest. For incorporated by reference ) . example , to drive specific expression of a tagged ribosomal In certain embodiments regulatory sequences that may be protein ( for example eGFP tagged L10a ) in medium spiny used to control expression can be derived from , include but neurons, regulatory sequences from a medium spiny neuron are not limited to , the following animal transcriptional specific gene locus , for example a Drdla or Drd2 locus, can 10 control regions that exhibit tissue specificity and that have be utilized . In another example , to drive specific expression been utilized in cell culture and transgenic animals . of a tagged ribosomal protein (for example eGFP tagged Examples of transcriptional control regions are: elastase I L10a ) in motor neurons , regulatory sequences from a motor - gene control region , enolase promoter, insulin gene control specific gene locus, for example choline acetyl transferase region , immunoglobulin gene control region , mouse mam ( Chat) locus , can be utilized . Similarly , to drive specific 15 mary tumor virus control region , albumin gene control expression of a tagged ribosomal protein ( for example eGFP region , alpha- fetoprotein gene control region , alpha 1 - anti tagged L10a ) in cerebellar cells , regulatory sequences from trypsin gene control region , beta .- globin gene control a cerebellar cell -specific gene loci. For example loci from region , myelin basic protein gene control region , myosin Pcp2 , Neurodi , Grm2, Grp , Septin4 , or Aldh111 can be light chain - 2 gene control region , and gonadotropic releas utilized , to drive specific expression in Purkinje cells , gran - 20 ing hormone gene control region . In other embodiments , the ule cells , golgi neurons , unipolar brush cells , Bergmann glial gene sequence from which the regulatory sequence derives cells , and astrocytes, respectively . An entire locus, an entire can be protein kinase C , gamma, TH - elastin , Pax7 , Eph non - coding region of a locus, a portion of a locus , or a receptors , islet- 1 and Sonic hedgehog . modified locus can be utilized as the regulatory sequences to Nucleic acids to be used for regulating expression may drive expression of tagged ribosomal proteins in a geneti - 25 include all or a portion of the upstream and / or downstream cally targeted , cell subtype specific manner . and 5 ' and / or 3 ' regulatory sequences from the naturally The tagged ribosomal gene coding sequences may placed occurring or modified locus of a selected gene . The regula under the transcriptional control of some or all of the tory sequences preferably direct expression of the tagged regulatory sequences from a particular locus such that the ribosomal protein sequences in substantially the same pat tagged ribosomal gene is expressed in substantially the same 30 tern as the endogenous characterizing gene within transgenic expression pattern as the endogenously regulated gene found organism , or tissue derived therefrom . This would provide of in that locus in the transformed organism , or at least in an for any and all cis - and trans- acting regulatory sequences anatomical region or tissue of the organism (by way of and transcriptional units that would direct expression of the example , in the brain , spinal cord , heart , skin , bones, head , tagged ribosomal protein in an endogenous regulatory fash limbs, blood , muscle , peripheral nervous system , etc . of an 35 ion . A regulatory sequence can be 1 kb , 5 kb , 10 kb , 50 kb , animal) containing the population of cells to be marked by 100 kb , 200 kb , 250 kb , 500 kb or even 1 Mb in length . expression of the tagged ribosomal protein gene coding In certain embodiments , the nucleic acids encoding the sequences. By “ substantially the same expression pattern ” is molecularly tagged ribosomal proteins may be selectively meant that the tagged ribosomal protein gene coding expressed in random but distinct subsets of cells , as sequences are expressed in at least 50 % , 60 % , 70 % 80 % , 40 described in Feng et al. ( 2000 , Imaging neuronal subsets in 85 % , 90 % , 95 % , and about 100 % of the cells shown to transgenic mice expressing multiple spectral variants of express the endogenous characterizing gene by in situ GFP , Neuron 28 ( 1 ): 41 -51 , which is hereby incorporated by hybridization , PCR , other gene expression detection meth reference in its entirety ) . Using such methods , independently ods or functional methods familiar to those with skill in the generated transgenic lines may express the nucleic acids art . 45 encoding the molecularly tagged ribosomal proteins in a The regulatory sequence is , e . g ., a promoter or enhancer, unique pattern , perhaps pathway -specific , or circuit - specific , or any other cis or trans activating element or transcriptional and although spatially separated , would all incorporate iden unit of a characterizing gene . In some embodiments this tical regulatory elements . characterizing gene is endogenous to a host cell or host 3 ) Placement and Construction of Regulatory Sequences organism (or is an ortholog of an endogenous gene ) and is 50 and Transgenes expressed in a particular select population of cells of the In certain embodiments the nucleotide sequences encod organism . The regulatory sequence can be derived from a ing the tagged ribosomal protein product may replace all or human , rat, or mouse or any mammalian gene associated a portion of the characterizing gene coding sequences in a with specific neuronal , cellular , and metabolic pathways . In genomic clone / locus of the characterizing gene , leaving only certain embodiments , a non mammalian regulatory sequence 55 all or part of the characterizing gene regulatory non - coding can be utilized . Cis and trans, 5 ' and 3 ' regulatory sequences sequences . and transcriptional units from several known pathways are In other embodiments , the tagged ribosomal gene coding amenable for cell - type specific , cell subtype - specific , devel- sequences are inserted into or replace transcribed coding or opmental stage -specific , temporal- specific , tissue -specific , non - coding sequences of the genomic characterizing gene pathway - specific , or circuit -specific expression of trans - 60 sequences , for example , into or replacing a region of an exon genes. Examples of known pathways include but are not or of the 3 ' UTR of the characterizing gene genomic limited to the adrenergic or noradrenergic neurotransmitter sequence . pathway, the dopaminergic neurotransmitter pathway, the In one embodiment, the tagged ribosomal protein gene GABAergic neurotransmitter pathway, the glutaminergic coding sequence is inserted into or replaces a portion of the neurotransmitter pathway , the glycinergic neurotransmitter 65 3 ' untranslated region (UTR ) of the characterizing gene pathway , the histaminergic neurotransmitter pathway , a neu genomic sequence . In another embodiment, the coding ropeptidergic neurotransmitter pathway , a serotonergic neu sequence of the characterizing gene is mutated or disrupted US 9 ,816 ,096 B2 21 22 to abolish characterizing gene expression from the nucleic example , a conditionally expressible nucleic acid of the acid construct without affecting the expression of the tagged invention can be created in which the coding region for the fusion protein gene . In certain embodiments , the tagged tagged ribosomal fusion protein gene ( and , optionally also ribosomal fusion protein gene coding sequence has its own the characterizing gene ) is operably linked to a genetic internal ribosome entry site ( IRES ) . 5 switch , such that expression of the tagged ribosomal fusion In some embodiments , the tagged ribosomal protein gene protein gene can be further regulated . One example of this coding sequences are inserted using 5 ' direct fusion wherein type of switch is a tetracycline -based switch . the tagged ribosomal protein gene coding sequences are in several embodiments , the nucleic acids of the invention inserted in - frame adjacent to the initial ATG sequence ( or comprise all or a significant portion of the genomic sequence adjacent the nucleotide sequence encoding the first two , 10 of the characterizing gene locus , at least all or a portion of three , four, five , six , seven or eight amino acids of the the 5 ' and /or 3' regulatory sequences of the characterizing characterizing gene protein product ) of the characterizing endogenously regulated gene , at least sufficient sequence 5 ' gene , so that translation of the inserted sequence produces a and / or 3 ' of the characterizing gene coding sequence to fusion protein of the first methionine ( or first few amino direct expression of the tagged ribosomal fusion protein acids ) derived from the characterizing gene sequence fused 15 gene coding sequences in the same expression pattern ( tem to the tagged ribosomal fusion protein gene protein . poral and/ or spatial ) as the endogenous counterpart of the In other embodiments , a tagged ribosomal fusion protein characterizing gene . In certain embodiments , the nucleic gene is inserted into a separate cistron in the 5 ' region of the acid of the invention comprises at least one exon , at least two characterizing gene genomic sequence and has an indepen - exons, at least three exons, all but one exon , or all but two dent IRES sequence . In certain embodiments , an IRES is 20 exons , of the characterizing endogenously regulated gene . operably linked to the tagged ribosomal fusion protein gene 4 ) Expression Using a Binary System coding sequence to direct translation of the tagged fusion Since the level of expression of the tagged ribosomal protein gene. The IRES permits the creation of polycistronic protein within a cell may needed in the efficiency of the mRNAs from which several proteins can be synthesized isolation procedure, in certain embodiments of the inven under the control of an endogenous transcriptional regula - 25 tion , a binary system can be used , in which the endogenous tory sequence. Such a construct is advantageous because it regulatory sequence such as a promoter drives expression of allows marker proteins to be produced in the same cells that a protein that then activates a second expression construct. express the endogenous gene (Heintz , 2000 , Hum . Mol . This second expression construct uses a strong regulatory Genet. 9 ( 6 ) : 937 - 43 ; Heintz et al. , WO 98 /59060 ; Heintz et sequence such as a promoter to drive expression of the al ., WO 01/ 05962 ; which are incorporated herein by refer - 30 tagged ribosomal fusion protein at higher levels than is ence in their entireties ) . possible using the endogenous regulatory region itself . In certain embodiments , exogenous translational control In certain embodiments , a particular population -specific signals , including , for example , the ATG initiation codon , gene drives expression of a molecular switch ( e . g . , a recom can be provided by the characterizing gene or some other binase, a transactivator ) in a population -specific manner. heterologous gene. The initiation codon is usually in phase 35 This switch then activates high - level expression though a with the reading frame of the desired coding sequence of the second regulatory element regulating expression of the tagged ribosomal fusion protein gene to ensure translation of tagged ribosomal protein . the entire insert. These exogenous translational control sig . For example , the molecularly tagged ribosomal protein nals and initiation codons can be of a variety of origins, both coding sequence may be expressed conditionally , through natural and synthetic . The efficiency of expression may be 40 the activity of a molecular switch gene which is an activator enhanced by the inclusion of appropriate transcription or suppressor of gene expression . In this case , the second enhancer elements , transcription terminators , etc . ( see gene encodes a transactivator , e . g ., tetr , a recombinase , or Bittner et al. , 1987 , Methods in Enzymol. 153 : 516 -44 ) . FLP, whose expression is regulated by the characterizing The construct can also comprise one or more selectable gene regulatory sequences . The gene encoding the molecu markers that enable identification and / or selection of recom - 45 larly tagged ribosomal protein is linked to a conditional binant vectors . The selectable marker may be the tagged element, e . g ., the tet promoter, or is flanked by recombinase ribosomal fusion protein gene product itself or an additional sites, e . g ., FRT sites , and may be located any where within selectable marker not necessarily tied to the expression of the genome. In such a system , expression of the molecular the characterizing gene. switch gene, as regulated by the characterizing gene regu In certain embodiments where a tagged ribosomal fusion 50 latory sequences, activates the expression of the molecularly protein gene is expressed conditionally , the tagged ribo - tagged ribosomal protein . somal fusion protein gene coding sequence is embedded in 5 ) Transcriptional Regulation Systems the genomic sequence of the characterizing gene and is In certain embodiments , the tagged ribosomal fusion inactive unless acted on by a transactivator or recombinase , protein gene can be expressed conditionally by operably whereby expression of the tagged ribosomal fusion protein 55 linking at least the coding region for the tagged ribosomal gene can then be driven by the characterizing gene regula fusion protein gene to all or a portion of the regulatory tory sequences. In such a system , expression of the trans sequences from the characterizing gene ' s genomic locus , activator gene, as regulated by the characterizing gene and then operably linking the tagged ribosomal fusion regulatory sequences, activates the expression of the tagged protein gene coding sequences and characterizing gene fusion protein gene. 60 sequences to an inducible or repressible transcriptional In a specific embodiment, a nucleic acid of the invention regulation system . Transactivators in these inducible or is expressed conditionally , using any type of inducible or repressible transcriptional regulation systems are designed repressible system available for conditional expression of to interact specifically with sequences engineered into the genes known in the art , e . g . , a system inducible or repres - vector . Such systems include those regulated by tetracycline sible by tetracycline (“ tet system ” ) ; interferon ; estrogen , 65 (“ tet systems” ) , interferon , estrogen , ecdysone , Lac opera ecdysone , or other steroid inducible system ; Lac operator, tor, progesterone antagonist RU486 , and rapamycin progesterone antagonist RU486 , or rapamycin ( FK506 ) . For (FK506 ) with tet systems being particularly preferred ( see , US 9 ,816 ,096 B2 23 24 e. g. , Gingrich and Roder , 1998 , Annu . Rev. Neurosci . 21: 6 ) Sources of Nucleic Acids 377 - 405 ; incorporated herein by reference in its entirety ). Nucleic acids comprising the characterizing gene These drugs or hormones (or their analogs ) act on modular sequences and tagged ribosomal fusion protein gene coding transactivators composed of natural or mutant ligand bind sequences can be obtained from any available source. In ing domains and intrinsic or extrinsic DNA binding and 5 most cases , all or a portion of the characterizing gene transcriptional activation domains . In certain embodiments, sequences and / or the tagged ribosomal fusion protein gene expression of the detectable or selectable marker can be coding sequences are known , for example , in publicly regulated by varying the concentration of the drug or hor - available databases such as GenBank , UniGene and the mone in medium in vitro or in the diet of the transformed Mouse Genome Informatic (MGI ) Database to name just a organism in vivo . 10 few , or in private subscription databases . With a portion of The inducible or repressible genetic system can restrict the sequence in hand , hybridization probes can be designed the expression of the detectable or selectable marker either using highly routine methods in the art ( for example filter temporally , spatially , or both temporally and spatially . hybridization or PCR amplification ) to identify clones con In other embodiments , expression of the tagged ribosomal taining the appropriate sequences for example in a library or fusion protein gene is regulated by using a recombinase 15 other source of nucleic acid . If the sequence of the gene of system that is used to turn on or off tagged ribosomal fusion interest from one species is known and the counterpart gene protein gene expression by recombination in the appropriate from another species is desired , it is routine in the art to region of the genome in which the marker gene is inserted design probes based upon the known sequence . The probes Such a recombinase system , in which a gene that encodes a hybridize to nucleic acids from the species from which the recombinase can be used to turn on or off expression of the 20 sequence is desired , for example , hybridization to nucleic tagged ribosomal fusion protein gene ( for review of tempo - acids from genomic or DNA libraries from the species of ral genetic switches and " tissue scissors ” using recombi- interest. By way of example and not limitation , genomic nases , see Hennighausen and Furth , 1999, Nature Biotech clones can be identified by probing a genomic DNA library nol. 17 : 1062 -63 ) . Exclusive recombination in a selected cell under appropriate hybridization conditions, e . g ., high strin type may be mediated by use of a site - specific recombinase 25 gency conditions , low stringency conditions or moderate such as Cre , FLP - wild type (wt ) , FLP - L or FLPe . Recom - stringency conditions, depending on the relatedness of the bination may be effected by any art -known method , e . g ., the probe to the genomic DNA being probed . method of Doetschman et al . ( 1987 , Nature 330 : 576 -78 ; 7 ) Vectors incorporated herein by reference in its entirety ) ; the method Briefly , the characterizing gene genomic sequences are of Thomas et al ., ( 1986 , Cell 44 : 419 - 28 ; incorporated herein 30 preferably in a vector that can accommodate lengths of by reference in its entirety ) ; the Cre - loxP recombination sequence ( for example , 10 kb ' s of sequence ) , such as system (Stemberg and Hamilton , 1981, J. Mol. Biol. 150 : cosmids , yeast artificial chromosomes ( YACs) and bacterial 467 -86 ; Lakso et al. , 1992 , Proc . Natl . Acad . Sci . USA 89 : artificial chromosomes (BACs ) , and encompass at least 50 , 6232 - 36 ; which are incorporated herein by reference in their 70 , 80 , 100 , 120 , 150 , 200 , 250 , 300 , 400 , 500 or 1000 kb entireties ) ; the FLP recombinase system of Saccharomyces 35 of sequence that comprises all or a portion of the charac cerevisiae ( O 'Gorman et al. , 1991 , Science 251 : 1351 - 55 ) ; terizing gene sequence. The larger the vector insert, the more the Cre -loxP - tetracycline control switch (Gossen and likely it is to identify a vector that contains the character Bujard , 1992 , Proc . Natl . Acad . Sci . USA 89 : 5547 -51 ) ; and izing gene cis and trans sequences of interest. In some ligand -regulated recombinase system (Kellendonk et al . , embodiments the vector containing the entire genomic 1999 , J. Mol. Biol. 285 : 175 - 82 ; incorporated herein by 40 sequence ( i. e . genomic locus) for the characterizing endog reference in its entirety ) . Preferably , the recombinase is enously regulated gene is present. In other embodiments the highly active , e . g ., the Cre - loxP or the FLPe system , and has entire genomic sequence cannot be accommodated by a enhanced thermostability (Rodriguez et al , 2000 , Nature single vector or such a clone is not available . In these Genetics 25 : 139 - 40 ; incorporated herein by reference in its instances (or when it is not known whether the clone entirety ) . 45 contains the entire genomic sequence ) , the vector contains at In certain embodiments, a recombinase system can be least the characterizing gene sequence with the start , i . e . , the linked to a second inducible or repressible transcriptional most 5 ' end , of the coding sequence in the approximate regulation system . For example , a cell -specific Cre - loxP middle of the vector insert containing the genomic mediated recombination system (Gossen and Bujard , 1992 , sequences and /or has at least 20 kb , 30 kb , 40 kb , 50 kb , 60 Proc . Natl. Acad . Sci . USA 89: 5547 -51 ) can be linked to a 50 kb , 80 kb , 100 kb or 200 kb of genomic sequence on either cell -specific tetracycline - dependent time switch detailed side of the start of the characterizing gene coding sequence . above (Ewald et al. , 1996 , Science 273 : 1384 - 1386 ; Furth et This can be determined by any method known in the art , for al. Proc . Natl. Acad . Sci. U . S . A . 91 : 9302 - 06 ( 1994 ) ; St- example , but not by way of limitation , by sequencing , Onge et al, 1996 , Nucleic Acids Research 24 ( 19 ): 3875 - 77 ; restriction mapping , PCR amplification assays, etc . which are incorporated herein by reference in their entire - 55 Once the appropriate vector containing the regulatory ties ). region / regulatory gene sequences has been created , the In one embodiment, an altered cre gene with enhanced tagged ribosomal fusion protein gene can be incorporated expression in mammalian cells is used (Gorski and Jones , into the characterizing gene sequence by any method known 1999 , Nucleic Acids Research 27 ( 9 ) : 2059 -61 ; incorporated in the art for manipulating DNA . In one embodiment, herein by reference in its entirety ) . 60 homologous recombination in bacteria is used for target In a specific embodiment, the ligand -regulated recombi- directed insertion of the tagged ribosomal fusion protein nase system of Kellendonk et al. ( 1999 , J . Mol. Biol. 285 : gene sequence into the genomic DNA encoding the charac 175 - 82 ; incorporated herein by reference in its entirety ) can terizing endogenously regulated gene and sufficient regula be used . In this system , the ligand -binding domain ( LBD ) of tory sequences to promote expression of the gene in its a receptor , e . g . , the progesterone or estrogen receptor, is 65 endogenous expression pattern , which characterizing gene fused to the Cre recombinase to increase specificity of the sequences have been inserted into a vector. The vector recombinase . comprising the tagged ribosomal fusion protein gene and US 9 ,816 ,096 B2 25 26 regulatory gene sequences is then introduced into the When entire genomic loci or portions thereof are used , few , genome of a potential founder organism for generating a line if any, site - specific expression problems are encountered , of transformed organisms, using methods well known in the unlike insertions of nucleic acids into smaller sequences . art , e . g . , those methods described herein . Such transformed When insertions of nucleic acids are too large , other prob organisms are then screened for expression of the tagged 5 lems can be encountered . In some embodiments , the vector ribosomal fusion protein gene coding sequences that mimics is a BAC containing genomic sequences into which a the expression of the endogenous characterizing gene . Sev - selected sequence encoding a molecular tag that has been eral different constructs containing nucleic acids of the inserted by directed homologous recombination in bacteria , invention may be introduced into several potential founder e . g ., by the methods of Heintz WO 98 /59060 ; Heintz et al. , organisms and the resulting transformed organisms are then 10 WO 01 /05962 ; Yang et al ., 1997 , Nature Biotechnol. 15 : screened for the best , ( e . g . , highest level ) and most accurate 859 - 865 ; Yang et al. , 1999 , Nature Genetics 22 : 327 - 35 ; (best mimicking expression of the endogenous characteriz which are incorporated herein by reference in their entire ing gene ) expression of the tagged ribosomal fusion protein ties. gene coding sequences. Using such methods , a BAC can be modified directly in The nucleic acid construct can be used to transform a host 15 a recombination -deficient E . coli host strain by homologous or recipient cell or organism using well known methods, recombination . i. e . , a cell that cannot independently support e . g ., those described herein . Transformation can be either a homologous recombination , e . g ., Rec A .sup . -. In one permanent or transient genetic change . In one aspect of the embodiment, homologous recombination in bacteria is used invention , a vector is used for stable integration of the for target- directed insertion of a sequence encoding a nucleic acid construct into the genome of the cell . In another 20 molecularly tagged ribosomal protein into the genomic aspect , the vector is maintained episomally . DNA encoding sufficient regulatory sequences ( termed 8 ) Bacterial Artificial Chromosomes (BACs ) and Other “ characterizing gene sequences " ) to promote expression of Vectors the tagged ribosomal protein in the endogenous expression The methods described herein provide transformed organ - pattern of the characterizing gene , which sequences have isms, e . g . , transgenic mice that express a tagged ribosomal 25 been inserted into the BAC . The BAC comprising the tagged protein within a chosen cell type . In some embodiments , fusion protein sequence under the control/ regulation of a BAC -mediated recombination ( Yang , et al . , 1997 , Nat . Bio regulatory region comprising sequences from a genomic technol. 15 ( 9 ) :859 - 865 ) is used to create the transformed locus of the desired characterizing gene is then recovered organism . Such expression is achieved by using the endog - and introduced into the genome of a potential founder enous regulatory sequences and transcriptional units of a 30 organism for a line of transformed organisms . In one aspect , particular gene, wherein the expression of gene is a defining the particular nucleotide sequence that has been selected to characteristic of the chosen cell type ( as also described in undergo homologous recombination is contained in an inde PCT /US02 / 04765 , entitled “ Collections of Transgenic Ani- pendent origin based cloning vector introduced into or mal Lines ( Living Library ) " by Serafini, published as WO contained within the host cell, and neither the independent 02/ 064749 on Aug . 22 , 2002 , which is incorporated by 35 origin based cloning vector alone , nor the independent reference herein in its entirety ) . A collection of transgenic origin based cloning vector in combination with the host animals expressing tagged ribosomal proteins within a set of cell, can independently support homologous recombination chosen cell types is assembled , and this is often referred to ( e . g . , is RecA ) . The exact method used to introduce the as a BACarray collection . tagged ribosomal protein encoding sequence and to remove In another embodiment, a nucleic acid of the invention is 40 (or not) the RecA ( or other appropriate recombination inserted into a yeast artificial chromosome (YAC ) (Burke et enzyme ) will depend upon the nature of the BAC library al ., 1987 Science 236 : 806 - 12 ; and Peterson et al . , 1997 , used ( for example , the selectable markers present on the Trends Genet. 13 : 61 ) . A collection of transgenic animals BAC vectors ) and such modifications are within the skill in expressing tagged ribosomal proteins within a set of chosen the art . cell types or subtypes is assembled , and this is often referred 45 Once the BAC containing the characterizing gene regu to as a YACarray collection . latory sequences and molecularly tagged ribosomal protein In other embodiments , the nucleic acid of the invention is coding sequences in the desired configuration is identified , inserted into another vector developed for the cloning of it can be isolated from the host E . coli cells using routine large segments of mammalian DNA , such as a cosmid or methods and used to make transformed organisms as bacteriophage P1 ( Sternberg et al. , 1990 , Proc . Natl. Acad . 50 described within . Sci. USA 87 : 103 -07 ) . The approximate insert size is about Alternatively, the BAC can also be engineered or modi 30 -35 kb for cosmids and 100 kb for bacteriophage P1. fied by “ E - T cloning ,” as described by Muyrers et al ( 1999 , In another embodiment, the nucleic acid of the invention Nucleic Acids Res. 27 (6 ): 1555 -57 , incorporated herein by is inserted into a P - 1 derived artificial chromosome ( PAC ) reference in its entirety ) . Using these methods, specific DNA (Mejia et al ., 1997 , Retrofitting vectors for Escherichia 55 may be engineered into a BAC independently of the pres coli -based artificial chromosomes (PACs and BACs ) with ence of suitable restriction sites . This method is based on markers for transfection studies , Genome Res . 7 ( 2 ) : 179 - 86 ) . homologous recombination mediated by the recE and recT The maximum insert size is about 300 kb . proteins (“ ET -cloning ” ) ( Zhang et al. , 1998 , Nat . Genet . BACs and other vectors used in the methods described 20 ( 2 ): 123 - 28 ; incorporated herein by reference in its here often can accommodate , and in certain embodiments 60 entirety ) . comprise, large pieces of heterologous DNA such as Host Cells and Organisms Engineered to Express Molecu genomic sequences. Such vectors can contain an entire larly Tagged Ribosomes genomic locus , or at least sufficient sequences to confer 1 ) Introduction of Vectors into Host Cells endogenous regulatory expression pattern and to insulate the In one aspect , a vector containing the nucleic acid encod expression of coding sequences from the effect of regulatory 65 ing the regulatory sequences and the molecularly tagged sequences surrounding the site of integration of the nucleic fusion protein can be introduced transiently or stably into the acid in the genome to mimic better wild type expression . genome of a host cell. In another aspect , the vector can be US 9 ,816 ,096 B2 27 28 transiently transfected wherein it is not integrated , but is delivery can utilize lentivirus, retrovirus , adenovirus, herpes maintained as an episome . The terms " host cell ” and simplex virus, adeno associated virus , or any other virus “ recombinant host cell ” are used interchangeably herein . amenable to gene delivery . It is understood that such terms refer not only to the 3 ) Transgenic Organisms particular subject cell but to the progeny or potential prog - 5 Any model organism can be engineered to express a eny of such a cell. Because certain modifications may occur molecularly tagged ribosome using methods well known in the art . These can include but are not limited to non -human in succeeding generations due to either mutation or envi primates , mice, rats, sheep , dogs, cows, goats , chickens , ronmental influences, such progeny may not, in fact, be amphibians, fish , zebrafish , (Danio rerio ), flies (Drosophila identical to the parent cell , but are still included within the 10 melanogaster ) , worms (Caenorhabditis elegans ) , or yeast scope of the term as used herein . (or other fungi) . A transgenic animal is a non -human animal A host cell can be any prokaryotic (e .g ., bacterium such in which one or more of the cells of the animal includes a as E . coli ) or eukaryotic cell (e .g ., a cell from a yeast, plant, nucleic acid that is non - endogenous ( i . e ., heterologous ) and insect ( e . g . , Drosophila ) , amphibian , amniote , or mammal, is present as an extrachromosomal element in a portion of its to name but a few . In certain embodiments the host cell is a 15 cell or stably integrated into its germ line DNA ( i. e ., in the human cell , a rodent cell , a mouse cell , a rat cell , a genomic sequence of most or all of its cells ) . In certain mammalian cell , an immortalized cultured cell or primary embodiments a transgenic animal comprises stable changes human or rodent cell. In specific embodiments , the host cells to the germline sequence . Heterologous nucleic acid is are human or rodent embryonic stem cells , neuronal stem introduced into the germ line of such a transgenic animal by cells , hippocampal stem cells , hippocampal progenitor cells , 20 genetic manipulation of, for example , embryos or embryonic or partially differentiated pluripotent cells , or tumor cells or stem cells of the host animal. cancer cells (particularly circulating cancer cells such as The heterologous nucleic acid may integrate into the those resulting from leukemias and other blood system genome of the founder organism by random integration . If cancers ). Methods can utilize genetically engineered host random , the integration preferably does not knock out, e . g . , cells that contain any of the foregoing tagged ribosomal 25 insert into , an endogenous gene( s ) such that the endogenous protein coding sequences, optionally operatively associated gene is not expressed or is mis -expressed with a regulatory element (preferably from a characterizing In other embodiments , the nucleic acid of the may inte gene, as described above ) that directs the expression of the grate by a directed method , e . g ., by directed homologous coding sequences in the host cell . Both cDNA and genomic recombination (“ knock -in " ) , Chappel , U . S . Pat. No . 5 , 272 , sequences can be cloned and expressed . 30 071 ; and PCT publication No. WO 91 / 06667 , published In one aspect , the host cell is recombination deficient, i. e ., May 16 , 1991 ; U .S . Pat. No. 5, 464 , 764 ; Capecchi et al. , Rec. sup . -, and used for BAC recombination . In specific issued Nov . 7 , 1995 ; U .S . Pat. No. 5 ,627 , 059 , Capecchi et al embodiments the host cell may contain more than one type issued , May 6 , 1997 ; U . S . Pat . No. 5 , 487 , 992 , Capecchi et of ribosomal fusion , where the fusion of the different ribo - al. , issued Jan . 30 , 1996 ) . somal is to the same or different tags . Alternatively , a single 35 Transformed organisms, e . g . , transgenic animals , can be ribosomal may be fused to more than one tag , for example , generated by random integration of a vector into the genome at both is N - terminal and C - terminal ends. of the organism , for example , by pronuclear injection in an A vector containing a nucleotide sequence can be intro - animal zygote as described above . Other methods involve duced into the desired host cell by methods known in the art , introducing the vector into cultured embryonic cells, for e . g . , transfection , transformation , transduction , electropora - 40 example ES cells using methods of injection or electropo tion , infection , microinjection , cell fusion , DEAE dextran , ration well known in the art , and then introducing the calcium phosphate precipitation , liposomes , LIPOFEC - transformed cells into animal blastocysts , thereby generating TINTM , lysosome fusion , synthetic cationic lipids, use of a a " chimeras ” or “ chimeric animals ” , in which only a subset gene gun or a DNA vector transporter, such that the nucleo - of cells have the altered genome. Chimeras are often used tide sequence is transmitted to offspring in the line. For 45 for breeding purposes in order to generate the desired various techniques for transformation or transfection of transgenic animal. Animals having a heterozygous alteration mammalian cells , see Keown et al. , 1990 , Methods Enzy - are generated by breeding of chimeras . Male and female mol. 185: 527 -37 ; Sambrook et al ., 2001, Molecular Clon - heterozygotes are typically bred to generate homozygous ing , A Laboratory Manual, Third Edition , Cold Spring animals . Harbor Laboratory Press , N . Y . 50 Methods for generating transgenic animals via embryo In certain embodiments , the vector is introduced into a manipulation and microinjection , particularly animals such cultured cell. In other embodiments , the vector is introduced as mice , have become conventional in the art and are into a proliferating cell (or population of cells ) , e . g . , a tumor described , for example , in U . S . Pat. Nos . 4 ,736 , 866 and cell, a stem cell, a blood cell , a bone marrow cell, a cell 4 , 870 ,009 , 4 , 873 , 191 , in Hogan , Manipulating the Mouse derived from a tissue biopsy, etc . 55 Embryo , (Cold Spring Harbor Laboratory Press, Cold 2 ) Introduction of Vectors to Create Transgenic Animals Spring Harbor, N . Y . , 1986 ) and in Wakayama et al. , 1999 , Particular embodiments encompass methods of introduc - Proc . Natl. Acad . Sci. USA , 96 : 14984 - 89 . Similar methods tion of the vector containing the nucleic acid , using pronu - are used for production of other transgenic animals. clear microinjection into the mononucleus of a mouse A transgenic founder animal can be identified based upon embryo or infection with a viral vector comprising the 60 the presence of the nucleic acid introduced in its genome construct . Methods of pronuclear injection into mouse and / or expression of mRNA in tissues or cells of the embryos are well -known in the art and described in Hogan animals. A transgenic founder animal can then be used to et al. 1986 , Manipulating the Mouse Embryo , Cold Spring breed additional animals carrying the nucleic acid of. These Harbor Laboratory Press , New York , N . Y . and Wagner et al. , transgenic animals can further be bred to other transgenic U .S . Pat . No . 4 ,873 , 191, issued Oct. 10 , 1989 , herein 65 animals carrying other heterologous nucleic acids. Progeny incorporated by reference in their entireties . Viral methods harboring homologously recombined or integrated DNA in of inserting nucleic acids are known in the art . Targeted their germline cells can be used to breed animals in which US 9 ,816 ,096 B2 29 30 all cells of the animal contain the homologously recombined are used to detect the tag , such as with the use of optics DNA by germline transmission of the nucleic acid of inter based sorting or magnetics - based sorting . est . Transformed organisms that exhibit appropriate expres Clones of the non -human transgenic animals described sion (e . g . , detectable expression having substantially the herein can also be produced according to the methods 5 same expression pattern as the endogenous characterizing described in Wilmut et al. , 1997 , Nature 385 : 810 - 13 and gene in a corresponding non - transgenic organism or ana PCT Publication NOS . WO 97 / 07668 and WO 97 /07669 . tomical region thereof, i . e . , detectable expression in at least Once the transgenic mice are generated they may be bred and maintained using methods well known in the art . By 50 % , 60 % , 70 % , 80 % , 90 % or, preferably , 95 % of the cells way of example , the mice may be housed in an environ - 10 shown to express the endogenous gene by in situ hybrid mentally controlled facility maintained on a 10 hour dark : 14 ization ) are selected as lines of transformed organisms. hour light cycle . Mice are mated when they are sexually BACarray Lines mature ( 6 to 8 weeks old ) . In certain embodiments , the In someembodiments , a collection of lines of transformed transgenic founders or chimeras are mated to an unmodified organisms that contain a selected subset of cells or a cell animal. In one embodiment, the transgenic founder or chi- 15 population expressing molecularly - tagged ribosomes is pro mera is mated to C57BL / 6 mice ( Jackson Laboratories ). In vided . In some aspects , when the transformed organism has a specific embodiment where the nucleic acid is introduced been created utilizing BAC - related methods , this is referred into ES cells and a chimeric mouse is generated , the chimera to as a BACarray collection . The collection comprises at is mated to 129 / Sv mice , which have the same genotype as least 2 individual lines, preferably at least 5 , 10 , 25 , 50 , 100 , the embryonic stem cells . Protocols for successful creation 20 500 , 1000 , 1500 , 2000 , 2500 , 5000 , 10 ,000 , 15 ,000 , or and breeding of transgenic mice are known in the art 30 , 000 individual lines. Each individual line is selected for (Manipulating the Mouse Embryo . A Laboratory Manual, the collection based on the identity of the subset of cells in 2nd edition . B . Hogan , Beddington , R ., Costantini, F . and which the molecularly tagged ribosomes are expressed . Lacy , E . , eds . 1994 . Cold Spring Harbor Laboratory Press : In one embodiment, a BACarray line related to neuronal Plainview , N . Y . ) . 25 diseases (neurodegenerative , neuropsychiatric , behavioral, Methods to establish heterozygousity or homozygousity etc ) can be created . These include , but are not limited to are well known in the art and utilize PCR and Southern BACarray lines specific for expression for cells involved in blotting. disease processes such as depression , obesity , anxiety, epi In some embodiments , the transgenic mice are so highly inbred to be genetically identical except for sexual differ - 30 lepsy , sleep , Parkinson ' s disease , ADHD , Huntington ' s dis ences . The homozygotes are tested using backcross and ease , addiction , dementia , Alzheimer' s disease , ALS , and intercross analysis to ensure homozygosity . Homozygous the like. For example , see FIG . 1 for a representative lines for each integration site in founders with multiple BACarray collection specific to central nervous system integrations are also established . Brother / sister matings for diseases . 20 or more generations define an inbred strain . In another 35 In one embodiment , a BACarray collection relating to preferred embodiment, the transgenic lines are maintained sub -regions of a tissue, for example regions of the central as hemizygotes . nervous system can be created . These include , but are not In an alternative embodiment, individual genetically limited to BACarray lines specific for expression in only altered mouse strains are also cryopreserved rather than striatal cells ( for example expression in medium spiny propagated . Methods for freezing embryos for maintenance 40 neurons, striatonigral and /or striatopallidal neurons ), in only of founder animals and transgenic lines are known in the art. cerebellar cells ( for example expression in Purkinje neurons , Methods for reconstituting frozen embryos and bringing the astrocytes , Bergmann glia , granule cells , etc ) , in only hip embryos to term are known in the art . pocampal cells ( for example , expression in CA1 neurons , The nucleic acid encoding the molecularly tagged ribo - CA2 neurons, CA3/ 4 neurons, glia , astrocytes , DG neurons ) , somal protein may be introduced into the genome of a 45 in only hypothalamic cells , in only spinal cord cells , in only founder plant (or embryo that gives rise to the founder plant) pineal gland cells , and the like . using methods well known in the art (Newell , 2000 , Plant In another related embodiment, a BACarray collection transformation technology. Developments and applications , relating to a particular biological function can be created . Mol. Biotechnol. 16 ( 1 ): 53 -65 ; Kumar and Fladung , 2001 , These include but are not limited to BACarray lines specific Controlling transgene integration in plants , Trends in Plant 50 for functions related to myelination , excitatory neural trans Science 6 ( 4 ) : 155 - 159 ) . The nucleic acid encoding the mission , motor function , migration , adhesion , infiltration , molecularly tagged ribosomal protein may be introduced processing of noxious stimuli , processing of sensory stimuli , into the genome of bacteria and yeast using methods visual processing , auditory processing , olfactory processing , described in Ausubel et al. , 1989 , Current Protocols in vestibular control, regulation of feeding and satiety , regula Molecular Biology , Green Publishing Associates and Wiley 55 tion of wakefulness and sleep , regulation of reward behavior Interscience , N . Y . , Chapters 1 and 13 , respectively ) . ( for example as how it relates to addictive behavior) , and the 4 ) Screening for Expression of Tagged Ribosomal Pro - like . teins in Transgenic Organisms Analyses of mRNA Species Potential founder organisms for a line of transformed The embodiments described herein provide for translation organisms can be screened for expression of the molecularly 60 profiling and molecular phenotyping of particular cells and / tagged fusion protein coding sequence by ribosomes in the or tissues . mRNA complexed in polysomes with tagged population of cells characterized by expression of the endog - ribosomal proteins are isolated and can be analyzed by any enous characterizing gene . method known in the art. In one aspect, the translational In one embodiment, immunohistochemistry using an anti - profile of cells expressing the tagged ribosomal proteins can body specific for the molecular tag or a marker activated or 65 be analyzed by isolating the mRNA and constructing cDNA repressed thereby is used to detect expression of the molecu - libraries or by labeling the RNA for gene expression analy lar tag . In another embodiment, other methods and devices sis , for example by disposing the mRNA on a microarray . US 9 ,816 ,096 B2 31 32 Embodiments of this invention utilize techniques described is incorporated by reference herein ). The library may be in US 2005 /0009028 , which is herein incorporated in its normalized and presented in a high density array , such as a entirety . microarray . In one aspect , mRNA bound by the tagged ribosomal In a particular embodiment , a subpopulation of cells proteins may be used to produce a cDNA library and , in fact, 5 expressing a molecularly tagged ribosomal protein is iden a collection of such cell type - specific, cell - subtype specific , tified and /or gene expression analyzed using the methods of tissue - specific , organism - specific , disease -specific , func tion -specific , cDNA libraries may be generated from differ Serafini et al. , WO 99 /29877 entitled “ Methods for defining ent populations of isolated cells . Such cDNA libraries are cell types ,” which is hereby incorporated by reference in its useful to analyze gene expression , isolate and identify cell 10 entirety . type -specific genes, splice variants and non -coding RNAs, Data from such analyses may be used to generate a as well as identify co -regulated gene sets for a particular cell database of gene expression analysis for different popula related to a function or a disease state . In another aspect, tions of cells in the animal or in particular tissues or such cell -type specific libraries prepared from mRNA bound anatomical regions, for example , in the brain . Using such a by, and isolated from , the tagged ribosomal proteins from 15 database together with bioinformatics tools, such as hierar treated and untreated or transgenic or otherwise manipulated chical and non -hierarchical clustering analysis and principal cells / animals having can be used , for example in subtractive components analysis , cells are " fingerprinted " for particular hybridization procedures , to identify genes expressed at indications from healthy and disease -model animals or tis higher or lower levels in response to a particular treatment sues, co -regulated gene sets for a particular function , and the or in a disease state as compared to untreated animals . The 20 like . mRNA isolated from the tagged ribosomal proteins may also Applications and Considerations be analyzed using particular microarrays generated and 1 ) Sensitivity analyzed by methods well known in the art. Gene expression The methods provided herein are applicable to any cell, analysis using microarray technology is well known in the tissue, or organism as well as any disease or disorder. The art . Methods for making microarrays are taught , for 25 sensitivity afforded by the ability to interrogate an individual example , in U . S . Pat. No. 5 ,700 ,637 by Southern , U . S . Pat . cell subtype in a complex heterogeneous tissue or organ No. 5 ,510 ,270 by Fodor et al. and PCT publication WO system provides a resolution than can be greater than with 99 /35293 by Albrecht et al ., which are incorporated by other techniques such as whole - tissue microarrays and in reference in their entireties. By probing a microarray with situ hybridization . In some embodiments the TRAP method various populations ofmRNAs , transcribed genes in certain 30 of detecting one ormore cell subtype - specific mRNAs/ genes cell populations can be identified . Moreover, the pattern of allows for identification of greater than 5 % , 10 % , 15 % , gene expression in different cell types of cell states may be 20 % , 25 % , 30 % , 35 % , 40 % , 45 % and even 50 % of the readily compared . mRNAs / genes enriched in that cell type which are not The mRNA bound by the tagged ribosomal proteins may detectable by other means such as whole - tissue microarrays be analyzed , for example by northern blot analysis , PCR , 35 or in situ hybridization . RNase protection , etc ., for the presence ofmRNAs encoding 2 ) Diseases and Disorders certain protein products and for changes in the presence or The methods provided herein are applicable to any cell , levels of these mRNAs depending on manipulation . tissue , or organism as well as any disease or disorder. In yet another embodiment, specific cells or cell popula - Several disease are cited in , but not limited to those found tions that express a potential a molecularly tagged ribosomal 40 in the ' The Merck Manual of Diagnosis and Therapy ' , often protein are isolated from the collection and analyzed for called simply ‘ The Merck Manual' ( 2006 ) . The diseases and specific protein -protein interactions or an entire protein disorders can be central nervous system disorders , periph profile using proteomics methods known in the art , for eral nervous system disorders , and non nervous system example , chromatography, mass spectroscopy, 2D gel analy - disorders . sis , etc . 45 Examples of neurodegenerative diseases /disorders Other types of assays may be used to analyze the cell include , but are not limited to : alcoholism , Alexander ' s population expressing the molecularly tagged ribosomal disease , Alper ' s disease , Alzheimer' s disease , Amyotrophic protein either in vivo , in explanted or sectioned tissue or in lateral sclerosis , Ataxia telangiectasia , Batten disease ( also the isolated cells , for example , to monitor the response of the known as Spielmeyer - Vogt- Sjogren -Batten disease ), Bovine cells to a certain manipulation / treatment or candidate agent 50 spongiform encephalopathy (BSE ), Canavan disease , Coc ( for example , a small molecule , an antibody, a hybrid kayne syndrome, Corticobasal degeneration , Creutzfeldt antibody , an antibody fragment, a siRNA , an antisense Jakob disease, Huntington ' s disease , HIV - associated RNA , an aptamer, a protein , or a peptide ) or to compare the dementia , Kennedy ' s disease , Krabbe ' s disease , Lewy body response of the animals , tissue or cells to expression of the dementia , Machado - Joseph disease ( Spinocerebellar ataxia target or inhibitor thereof, with animals , tissue or cells from 55 type 3 ) , Multiple sclerosis , Multiple System Atrophy, Nar animals not expressing the target or inhibitor thereof. The colepsy, Neuroborreliosis , Parkinson ' s disease , Pelizaeus cells may be monitored , for example , but not by way of Merzbacher Disease , Pick 's disease, Primary lateral sclero limitation , for changes in electrophysiology , physiology ( for sis , Prion diseases , Refsum ' s disease , Sandhoffs disease , example , changes in physiological parameters of cells , such Schilder' s disease, Subacute combined degeneration of spi as intracellular or extracellular calcium or other ion concen - 60 nal cord secondary to Pernicious Anaemia , Schizophrenia , tration , change in pH , change in the presence or amount of Spielmeyer - Vogt- Sjogren -Batten disease (also known as second messengers , cell morphology , cell viability , indica - Batten disease ), Spinocerebellar ataxia (multiple types with tors of apoptosis , secretion of secreted factors , cell replica varying characteristics ) , Spinal muscular atrophy, Steele tion , contact inhibition , etc .) , morphology , etc . Richardson - Olszewski disease , and Tables dorsalis . In particular embodiments , the isolated mRNA is used to 65 Examples of neuropsychiatric diseases/ disorders include , probe a comprehensive expression library ( see , e . g . , Serafini but are not limited to : depression , bipolar disorder , mania , et al. , U . S . Pat. No. 6 , 110 ,711 , issued Aug . 29 , 2000 , which obsessive compulsive disease , addiction , ADHD , schizo US 9 ,816 ,096 B2 33 34 phrenia, auditory hallucinations, eating disorders, hysteria , that are translationally profiled in cholinergic motor neu autism spectrum disorders and personality disorders . rons. In this embodiment, the cholinergic motor neurons are Examples of neurodevelopmental diseases / disorders spatially separated . include , but are not limited to : attention deficit hyperactivity In another exemplary embodiment the cell type of interest disorder ( ADHD ) , attention deficit disorder (ADD ) , schizo - 5 are cerebellar neurons and glia , specifically Purkinje neu phrenia , obsessive - compulsive disorder (OCD ), mental rons and Bergmann glia . Ribosomal protein L10a fused to retardation , autistic spectrum disorders ( ASD ) , cerebral eGFP under the control of either a Pcp2 or Septin4 -specific palsy , Fragile - X Syndrome, Downs Syndrome, Rett ' s Syn - regulatory sequence is expressed and translational profiles drome, Asperger ' s syndrome, Williams- Beuren Syndrome, are obtained . Table 20 identifies genes that are translation childhood disintegrative disorder , articulation disorder, 10 ally profiled in cerebellar Purkinje neurons and Bergmann learning disabilities ( i. e ., reading or arithmetic ) , dyslexia , glia . expressive language disorder and mixed receptive -expres - In another embodiment a gene translational profile fol sive language disorder, verbal or performance aptitude . lowing the manipulation of a cell, tissue or organism is Diseases that can result from aberrant neurodevelopmental obtained . The method comprises manipulating a cell, tissue processes can also include , but are not limited to bipolar 15 or organism , using the TRAP methodology to obtain a disorders , anorexia , general depression , seizures, obsessive translational profile , and comparing the profiled to a refer compulsive disorder (OCD ) , anxiety, bruixism , Angleman 's ence profile from an non -manipulated cell, tissue or organ syndrome, aggression , explosive outburst, self injury , post ism . Manipulations include but are not limited to : traumatic stress , conduct disorders, Tourette ' s disorder , ste - a . Pharmacological: for example administration of a can reotypic movement disorder , mood disorder, sleep apnea , 20 didate agent such as a small molecule antagonist or an restless legs syndrome, dysomnias, paranoid personality agonist; administration of a pharmacological agent to disorder , schizoid personality disorder , schizotypal person recapitulate a disease such as the administration of ality disorder , antisocial personality disorder , borderline MPTP or OHDA to an animal model or cell culture to personality disorder, histrionic personality disorder , narcis induce a Parkinson ' s disease -like state ; or administra sistic personality disorder, avoidant personality disorder , 25 tion of a drug or substance of abuse , for example dependent personality disorder , reactive attachment disor cocaine or alcohol; der ; separation anxiety disorder ; oppositional defiant disor b . Genetic : for example introduction of a germline or der ; dyspareunia , pyromania , kleptomania , trichotillomania , non - germline mutation or transgene to recapitulate an gambling, pica , neurotic disorders, alcohol- related disor animal or cellular model of a disease or disorder such ders , amphetamine- related disorders , cocaine - related disor - 30 as ataxia , Parkinson ' s disease , Alzheimer ' s disease , ders , marijuana abuse , opioid - related disorders , phencycli autism spectrum disorders and the like ; dine abuse , tobacco use disorder, bulimia nervosa , c . Mechanical: for example surgical treatment ; and /or delusional disorder, sexual disorders , phobias , somatization d . Environmental: for example change of habitat, climatic disorder , enuresis , encopresis , disorder of written expres change , reversal of day -nite cycles ; for example induc sion , expressive language disorder, mental retardation , 35 tion of a chronic -mild -stress protocol, art - recognized , mathematics disorder , transient tic disorder, stuttering , to recapitulate a depression - like phenotype perturba selective mutism , Crohn ' s disease , ulcerative colitis , bacte tions . Examples of candidate agents are but not limited rial overgrowth syndrome, carbohydrate intolerance , celiac to a small molecule , an antibody , a hybrid antibody , an sprue , infection and infestation , intestinal lymphangiectasia , antibody fragment, a siRNA , an antisense RNA, an short bowel syndrome, tropical sprue , Whipple 's disease , 40 aptamer, a protein , or a peptide . In exemplary embodi Alzheimer ' s disease , Parkinson ' s Disease , ALS , spinal mus ments , the cell is a cell of a nervous system , such as a cular atrophies, and Huntington ' s Disease . Further neuronal or glial cell , a striatal cell , a cerebellar cell , a examples , discussion , and information on neurodevelop hippocampal cell, a hypothalamic cell, a cortical cell , a mental disorders can be found , for example , through the dopaminergic cell, a spinal cord cell. In other embodi Neurodevelopmental Disorders Branch of the National Insti- 45 ments the cell is a neuron such as a dopaminergic tute of Mental Health (worldwide website address at nihm neuron or a medium spiny neuron . .nih . gov / dptr /b2 - nd .cfm ). In an exemplary embodiment, the effect of cocaine on the 3 ) Profiling of mRNA Species translational profile of dopaminergic striatonigral and stri In one embodiment, the invention provides for a method atopallidal cells is obtained . In related embodiments the to obtain a translational profile of a cell type of interest . The 50 effects of acute cocaine administration and the effects of method comprises expressing a tagged ribosomal protein chronic cocaine administration is obtained . In specific under the control of a regulatory sequence specific to a gene embodiments , the gene translation changes following acute expressed in the cell type of interest, isolating mRNAs cocaine treatment comprise those identified in Table 15 . In complexed with the ribosomal protein from the cell , and related specific embodiments, the gene translation changes identifying the mRNAs, thereby obtaining a translational 55 following chronic cocaine treatment comprise those identi profile for the cell type of interest. fied in Table 16 . In one exemplary embodiment the cell types of interest 4 ) Disease Screening , Diagnostics, Prognostics, and are striatonigral and striatopallidal cells . L10a fused to eGFP Theranostics under the control of a Drdla or a Drd2 specific regulatory One embodiment of this invention is to establish a trans sequence is expressed and translational profiles are obtained . 60 lational profile and molecular phenotype for a tissue or cell Tables 10 , 13 , 17 , and 18 identify genes that are transla - type taken from a subject to be screened for, suspected of tionally profiled in striatonigral and striatopallidal cells . having , or presenting with a particular disease or disorder , In another exemplary embodiment the cell type of interest using the methods described herein . In this aspect , markers are cholinergic motor neurons . Ribosomal protein L10a associated with and / or indicative of a particular disease or fused to eGFP under the control of a choline acetyl trans - 65 disorder are identified . In one embodiment, in a manner ferase ( chat) -specific regulatory sequence is expressed and similar to a biopsy , a subject ' s tissue can be removed for translational profiles are obtained . Table 19 identifies genes sampling . In another embodiment, a sampling of cerebro US 9 ,816 ,096 B2 35 36 spinal fluid (CSF ) will be obtained from the subject . In other exemplary embodiment, the target of interest is Gpr6 . Gpro embodiments , any bodily fluid can be obtained from the represents a target for which antagonists can be screened for subject. Following the isolation of desired tissue or fluid , a and developed as a therapeutic for Parkinson ' s disease , due molecularly tagged ribosomal protein is introduced with the to its overexpression . regulatory elements required for a cell- type specific expres - 5 In a similar manner , an mRNA found to be downregulated sion . The translational profile and molecular phenotype is in a cell type of interest associated with a disease or disorder , established using the methods described herein . The result as above , could represent a target for agonism . That is , the ing profile and phenotype is then be compared to a profile protein encoded by the identified mRNA would be a thera and phenotype obtained using said methods from a subject peutic target for which agonists could be developed . or subjects not having or presenting with the particular 10 7 ) Personalized Medicine disease or disorder, i. e . a reference cell , tissue or subject. In one embodiment, a method is provided for assessing The results can be used for diagnostics , prognostic , or whether a subject in need thereof is amenable to a thera theranostic purposes in relation to a disease or disorder. The peutic agent, comprising determining a translational profile disease or disorder to be detected , diagnosed , prognosed or for a cell type , cell sub - type or tissue from the subject, theranosed , can be any disease named herein , or previously 15 determining if the translational profile is predictive of treat undescribed diseases and disorders. ment with one or more therapeutic agents , and identifying In certain embodiments , BAC -mediated expression can the one or more therapeutic agents to administer to the be utilized to drive expression of a molecularly tagged subject. ribosomal protein in a particular cell type. Several BACar In another embodiment, a method is provided for screen rays have been created and collected that, based on expres - 20 ing one or more therapeutic agents in a subject in need sion of the endogenously regulated gene , have restricted thereof comprising , administering one or more therapeutic patterns of expression that are directly relevant to several agents to a subject, determining a translational profile for a diseases or disorders ( FIG . 1 ) cell type , cell sub - type or tissue from the subject , comparing 5 ) Screening for Modulatory Agents the translational profile obtained to one or more reference Another embodiment of this invention is to screen for 25 profiles that indicate a positive or negative prognosis , and candidate agents for modulation of a desired modulatory determining the treatment should continue or be modified (antagonistic , agonistic , synergistic modulation ) activity . In based on the comparison . In a further embodiment, it is certain embodiments a vehicle or candidate agent is admin - determined that a different treatmentmodality ( e . g ., different istered in a single or repeated dose to either a cell type or a therapeutic agent should be administered , such as for subject. Following administration a translational profile is 30 example , a second line drug , that is shown to be amenable established for the cell type of interest based on the methods to the translational profile observed for a first line drug ) . disclosed herein . The profile and phenotypes from those 8 ) Co -regulated Gene Sets dosed with candidate agent are compared and related to the In one embodiment, a method to identify a co - regulated profile and phenotype from those dosed with a vehicle ( i . e . gene set for a known function or a candidate function for a reference sample ) . A determination is made whether a can - 35 novel gene is provided . The method comprises determining didate agentmodulates translation and phenotype of one or the translational profiles for a plurality of cell types that more mRNA isolated from the cell type of interest , thereby express a gene associated with a specific function , compar screening said candidate agent for translational modulation . ing the translational profiles to determine what additional In some embodiments the candidate agent may be a candi- genes are similarly regulated thereby identifying a co date therapeutic agent or drug . In other embodiments , the 40 regulated gene set involved in the function . candidate agent may be a toxin . In some embodiments the In further embodiments the function can be a cellular candidate agent may be but is not limited to a small function or a cellular process. In other related embodiments molecule , an antibody , hybrid antibody or antibody frag - the method is applied to determine a co - regulated gene set ment, a siRNA , an antisense RNA , an aptamer , a protein involved in myelination , excitatory neural transmission , therapeutic , or a peptide . 45 motor function , cellular migration , cellular adhesion , cellu 6 ) Therapeutic Target Screening and Selection lar infiltration , processing of noxious stimuli , processing of Another aspect of this invention is to identify candidate sensory stimuli , visual processing , auditory processing, therapeutic targets for a disease or disorder. In certain olfactory processing , vestibular control, regulation of feed embodiments this would entail transforming a cell or cre - ing and satiety , regulation of wakefulness and sleep , or the ating an organism expressing a molecularly tagged ribo - 50 regulation of reward behavior. somal protein and exposing the cell or organism to a In one exemplary embodiment, the method is applied to perturbation or stimulus, mRNA transcripts selectively determine a co -regulated gene set involved in myelination down regulated or up regulated would be potential targets comprising determining the translation profiles for a plural for ameliorating the perturbation . In other embodiments , a ity of cells that express the myelin basic protein (Mbp ). In translational profile from a cell from a subject with a disease 55 a related embodiment the gene set comprises one or more of or disorder , a cell that has been perturbed , etc will be the genes listed in Table 9 . compared to an otherwise normal or unperturbed cell . The This method can be useful in identifying gene sets for differential translational profile will allow for identification known functions or candidate functions for novel gene of potential therapeutic targets , products , since in many cases the cohorts of co - regulated In one embodiment, any mRNA found to be upregulated 60 genes can include genes with well known functions in a cell type of interest associated with a disease or disorder, Kits for example in but not limited to a striatal, hippocampal, In a further aspect, the present invention provides kits . In cortical, cerebellar, spinal cord , hypothalamic , pineal, reti- certain embodiments the kit contains reagents for determin nal , auditory , olfactory , vestibular, or brain stem cell may i ng the presence , absence , and / or differential presence , of represent a target for antagonism . That is , the protein 65 one or more markers indicative of a disease , disorder, and / or encoded by the identified mRNA would be a therapeutic pharmacologic , genetic , or environmental manipulation . The target for which antagonists could be developed . In one disease could be , but is not limited to a neurodegenerative , US 9 ,816 ,096 B2 37 38 neuropsychiatric , neurodevelopmental disorder in a sample Example 1 from an individual suspected of having a susceptibility to such a disease or disorder. In another embodiment , the Molecular Tagging of a Ribosomal Proteins disease or disorder is a proliferative disease such as a cancer. 5 In other embodiments the kit is utilized to identify co mRNAs translated into protein are at one point usually regulated gene sets for a particular biological function . attached to a ribosome or a polyribosome complex (poly somes ) . Any tag , not limited to those named herein , fused to Biological functions are described herein . a ribosomal protein allows for isolation of bound mRNAS . In one embodiment, the kit contains a customized set of 10 eGFP fused to the N -terminus of the large subunit ribosomal clones , vectors, molecular tags from which to choose , for protein L10a, hereafter eGFP -L10a , was utilized in this and molecular labeling and materials such as CDs, instructions ensuing examples, but is by no ways limiting to the tens , hundreds, thousands, or even more tag -protein combinations for use , and other reference guides that would allow the that can be utilized . individual to choose the correct clone/ vector for the cell 15 FIG . 2 : (a ) A schematic is presented to illustrate affinity type , tissue, molecular pathway, or circuit of choice . In purification of eGFP -tagged Polysomes (originating from another embodiment the kit contains a recombinant vector the target cell population ) using anti -GFP antibody - coated encoding a nucleic acid sequence which encodes a ribo beads (Schematic in a ). Fusions of Enhanced Green Fluo somal protein and a detectable tag operably linkedauto to a 20 rescent Protein ( eGFP ) with ribosomal proteins were regulatory region . In particular embodiments , the kit can screened for efficient incorporation into polysomes to pro contain a customized BACarray clone collection relevant for vide a tag for all translated cellular mRNAs. ( b ) : A BAC a particular disease or disorder . carrying the eGFP -L10a fusion protein was transfected into In one embodiment a kit includes a recombinant vector 25 HEK293T cells . The eGFP -L10a fusion protein ' s nucleolar engineered to express a nucleic acid sequence encoding a ribosomal protein and a detectable tag operably linked to a and cytoplasmic localization was consistent with incorpo regulatory region containing sequences endogenous to a ration into intact ribosomes and immuno -electron micros genomic locus of a gene of interest. In a related embodiment copy data demonstrated its presence on polysome com the ribosomal protein is fused in - frame to a detectable tag . 30 plexes . The figure displays transmission electron In certain embodiments , the recombinant vector is a BAC . micrographs of anti -GFP coated magnetic beads after incu The gene of interested can be selected from but is not limited bation with extracts taken from HEK293T cells transfected to one that expresses in a striatal cell , cerebellar cell , cortical with an empty vector ( left panel) or the eGFP - L1010a cell, hypothalamic cell, hippocampal cell , brainstem cell , and a spinal cord cell . In exemplary embodiments the tag is 355 construct (right panel) ; images acquired at 50, 000x magni eGFP and the ribosomal protein is L10a . fication , inserts enlarged by a factor of 2 .3x . There was no gross alteration in cell physiology or growth EXAMPLES rate was evident in eGFP -L10a transfected cells , as seen by fluorescence microscopy of eGFP -L10a expression in cells . The methodology reported here provides an enabling 40 HEK293T cells grown on coverslips were transiently trans technology for translational profiling and molecular pheno - fected , grown for two days, fixed with paraformaldehyde , typing . Examples allow for defining the distinguishing and mounted with media containing 4 ', 6 -diamidino -2phe molecular characteristics of closely related or critical cell nylindo (DAPI ) to stain DNA . types. Examples also demonstrate that methods described herein can be employed to analyze physiological adaptations 45 Example 2 of specific cell types in vitro , in vivo , ex vivo , or in situ . In various embodiments , compositions and methods fea ture methodology , which readily and reproducibly identify Isolation , Purification and Analysis of translated mRNAs in any cell type or tissue of interest . This 50 Ribosome- mRNA Complexes In Vitro methodology involves expression of a molecularly tagged ribosomal transgene , which enables tagging of polysomes Rapid immunoaffinity purification of polysomes was for isolation , purification and identification of mRNA, in achieved from HEK293T cells transiently transfected with specific populations. In some embodiments , this can be the eGFP -L10a transgene but not mock transfected cells . achieved using Bacterial Artificial Chromosome (BAC ) 55 HEK293T cells transfected with eGFP -L1010a were transgenic mice, allowing translational profiling and homogenized in lysis buffer . The solubilized and clarified molecular phenotyping from whole animals . lysate was loaded onto a linear density ( 20 - 50 % w / w ) The following examples are offered to illustrate , but not gradient of sucrose and centrifuged for 2 hours at 4° C . using to limit the claimed invention . Some embodiments of the a Beckman SW41 rotor at 40 ,000 r . p . m . ( 200 , 000xg ) . 750 present invention have been shown and described herein , but 60 ul fractions were collected as absorbance at 254 nm was it will be obvious to those skilled in the art that such monitored with an ISCO UA - 6 UV detector. embodiments are provided by way of example only . Numer - Immunoaffinity purification of polysomes from trans ous variations, changes, and substitutions will now occur to fected cell cultures (in which approximately 30 % of cells those skilled in the art without departing from the invention . expressed eGFP - L10a ) gave an approximate 10 % overall It should be understood that various alternatives to the 65 co - purification of untagged ribosomal proteins and ribo embodiments of the invention described herein may be somal RNA , and led to the recovery of only translated employed in practicing the invention . mRNAs ( Table 3 ) . US 9, 816 ,096 B2 39 40 TABLE 3 mples; * indicates the presence of a light eGFP -L10a band upon longer exposure . No endogenous Rp110a or Rp17 was Total RNA yields from cultured cell BACarray purifications. HEK293T cells were transiently transfected with eGFP (mock recovered in the bound (IP ) fraction of mock samples, while transfection ) or eGFP -L10a constructs . Transfection efficiency was endogenous Rp110a and Rp17 were both recovered in the approximatelyximately 30 %% . Plates of different ssizes were used to grow mock 5 bound ( IP ) fraction of untreated and iron - treated samples . or eGFP - L10a -transfected cells, which is reflected in different The reduced recovery of endogenous Rpl10a versus endog input RNA amounts . Input and immunoprecipitated (bound ) RNA were purified and the quantity and purity of RNA were determined enous Rp17 in the immunoprecipitation likely reflects com using a Bioanalyzer 2100 (Agilent Technologies ) . petition between endogenous Rp110a and eGFP -L10a for incorporation into ribosomes . ( f ) The iron - induced fold Total RNA Total RNA Yield 10 change in Fth1 mRNA levels relative to Actb mRNA levels Transfection Bound (ng ) Input ( ng ) in samples immunoprecipitated ( IP ) from either polysomes eGFP (Mock ) 61 33 ,320 0 . 2 or from whole cell lysates ( range of fold -change in poly eGFP - L10a 6 , 117 56 ,630 10 . 8 some IP samples: 2 .01 - 2 .43 ; range of fold - change in direct IP samples : 1 . 80 - 1 . 93 ) was similar to the change observed in FIG . 3 displays immunoprecipitation of translated 15 the polysome gradient fraction before immunoprecipitation (c ) . mRNAs from transfected cells . HEK293T cells were tran Methods siently transfected ( ~ 30 % efficiency ) with eGFP (mock ) or Standard methods for immunoblotting were used . Anti eGFP -L10a constructs and grown in medium alone ( no bodies were used as follows : GFP detection : JL -8 , Clontech treatment, NT) or in medium supplemented with 100 ug /ml 20 (Mountain View . Calif ) . 1 : 2 . 000 in 5 % non - fat milk /PBST ferric ammonium citrate [ pH 7 . 0 ] ( iron ) for 36 hours . ( a ) (phosphate buffered saline - 0 . 05 % Tween - 20 ; Rp17 detec Induction of the iron -storage protein Ferritin was seen after tion : NB200 - 308 , Novus Biologicals (Littleton , Colo . ) , 1 : 2 , 36 hours of iron treatment. ( b ) The post- mitochondrial 000 in 5 % IgG - free bovine serum albumin / PBS- T ; Rp110a supernatants of untreated or iron -treated cells were loaded detection : H00004736 -M01 , Abnova Corporation (Taipei onto linear sucrose gradients ( 20 - 50 % w / w ) . After velocity 25 City , Taiwan ) , 1 : 2 ,000 in 5 % IgG - free bovine serum albu sedimentation , fractions (direction of sedimentation noted min /PBS - T; Ferritin detection : 65077 , MP Biomedicals (So by arrow ) were collected while UV absorbance (254 nm ) lon , Ohio ), 1 : 1 ,500 in 0 . 2 % l -block ( Applied Biosystems, was being measured . A representative trace is shown , as Foster City , Calif . )/ PBS - T ; B - Actin detection : Ab8224 , profiles from non - treated and iron - treated lysates looked Abcam (Cambridge , Mass . ) , 1 : 2 ,500 in 5 % nonfat milk / nearly identical. Non - polysome and polysome gradient frac - 30 PBS - T . tions were generated as indicated . To avoid Ferritin mRNAs associated with mRNPs, only heavier polysomes (with Example 3 greater than 4 ribosomes ) were included in the polysome fraction . ( c ) After iron treatment, as expected , a shift of Characterization and Analysis of BACarray Ferritin heavy chain mRNA ( Fth1 ) out of non - polysome 35 Transgenic Mice fractions into polysome fractions was observed , as deter mined by reverse transcription followed by quantitative PCR 1 ) Overview of total RNA purified from non - polysome and polysome Comparative analysis of BACarray data can provide gradient fractions ( range of fold -change in non -polysome important mechanistic insights into complex biological sys fraction : 0 . 16 - 0 . 22 ; range of fold -change in polysome frac - 40 tems. BACarray translational profiling permits comprehen tion : 1 . 83 - 2 . 15 ). (d ) Immunoprecipitations were performed sive studies of translated mRNAs in genetically defined cell from non - polysome or polysome gradient fractions and populations, and their responses to physiological perturba 0 .2 % of the Input (IN ), 0 .02 % of the unbound (UB ), and 1 % tions. To establish the generality of this approach , BACarray of the bound ( IP ) samples were loaded onto gels for immu translational profiles for twenty four distinct and diverse noblot analysis with eGFP or Rp17 antibodies; * indicates 45 CNS cell populations are presented here . Identification of the presence of a weak Rp17 band upon much longer cell -specific and enriched transcripts, previously not identi exposure . eGFP -L10a was recovered equally well from fied in whole tissue microarray studies are provided as non - polysome and polysome fractions with or without iron examples to illustrate the added value of comparative analy treatment. Rp17 was recovered equally well from untreated sis of these large datasets . The BAC transgenic strategy has or iron -treated polysome fractions. Rp17 was not present in 50 been applied (Heintz , 2004 ; Yang et al. , 1997 ) to provide the non - polysome fraction , presumably because , unlike the high resolution anatomical data and BAC vectors for the overexpressed eGFP - Rp110a , it was all incorporated into design of genetic studies of specific , morphologically polysomes. As expected from the lack of Rp17 (and thus defined cells in the CNS (Gong et al ., 2003 ) (www .gensa assembled ribosomes) in the non - polysome fraction , immu t .org ). Heiman et al. have reported the development of the noprecipitations from non -polysome fractions did not pull 55 BACarray translational profiling methodology for use in down any RNA above background , indicating that the discovery of the complement of proteins synthesized in any immunoprecipitation was specific to translated messages . ( e ) genetically defined cell population . Here, the methodology To determine if the translating ribosome affinity purification is used to generate BACarray transgenic mice for a wide ( TRAP ) methodology could faithfully reflect the changes variety of anatomically and genetically defined cell types in observed in c , a direct eGFP immunoprecipitation of post - 60 the mammalian brain . In some embodiments , such a collec mitochondrial supernatants ( whole cell lysates, unfraction - tion can provide a resource that will allow detailed molecu ated ) was performed . 0 . 5 % of input (IN ) , 0 . 5 % of the lar phenotyping of CNS cell types at specified developmen unbound fraction (UB ) , and 1 . 0 % of the bound ( IP ) samples tal stages, and in response to a wide variety of were loaded onto gels for immunoblot analysis with eGFP pharmacological, genetic or behavioral alterations. The mice ( top ) , Rp110a (middle ) , or Rp17 ( bottom ) antibodies . eGFP - 65 and data presented confirm the generality of the BACarray L10a was recovered equally well from untreated and iron approach and provide a resource for studies of the molecular treated samples , as was all eGFP from mock Sa - bases for cellular diversity in the mammalian brain . US 9 ,816 ,096 B2 41 42 The generation of BACarray translational profiles of by their large cell bodies and molecular layer dendrites , specific CNS cell types requires targeting of the eGFP - L10a granule cells by their small size , dense packing , and location ribosomal protein fusion to desired CNS cell types , affinity in the granule layer , and Bergmann glial cells by their purification of polysomal RNAs from these cell types , and morphology , radial projections , and close proximity to interrogation of the resultant mRNA populations using mas - 5 Purkinje cells . IHC analysis of eGFP -L10a fusion proteins sively parallel analytical techniques. To provide a resource in these well known cell types using these regulatory regions for comparative analysis of diverse CNS cell types , allow for identification of well known and previously described in this examples is : the application of this strategy unknown cell types . The expression of the eGFP -L10a to generate and characterize further BA?array transgenic transgene from each BAC driver is regionally correct, con lines , to isolate and characterize mRNA populations from 10 forming both to the literature and to the expectation given twenty four cell types targeted in these lines , to analyze these the anatomic data presented in GENSTAT and for these cell data relative to one another, and to archive and present the types the distribution of the eGFPL10a fusion protein , while BACarray transgenic lines and their associated anatomic and more limited than that of soluble eGFP , is sufficient to microarray data . provide enough anatomic detail to unambiguously identify 2 ) Selection of Drivers to Target Specific CNS Cell Types 15 these well described CNS cell types. To confirm the general applicability of the translating Many BACarray lines, the eGFP -L10a fusion protein is ribosome affinity purification ( TRAP ) methodology , and to detected in multiple structures within the brain . An example obtain initial data concerning the depth of information one is the characterization of fusion protein IHC in cholinergic can obtain with this approach across multiple cell types , cell populations targeted in the Chat BACarray lines . In this BACs reported by the Gene Expression Nervous System 20 case expression is clear in brainstem motor neurons, spinal Atlas (GENSAT ) Project (Gong et al, 2003 ; S . Falcon , R . cord motor neurons, neurons of the corpus striatum , basal Gentleman , Bioinformatics 23 , 257 - 8 (2007 ) ; both incorpo forebrain projection neurons , and neurons of the medial rated in their entirety ) were chosen to specifically target a habenula . As detailed below , BACarray translational profiles wide range of neurons and glia from different structures for four of these cholinergic cell populations were collected throughout the CNS . 25 by separately dissecting the spinal cord , brainstem , corpus 3 ) BACarray Mice striatum and basal forebrain prior to affinity purification of To genetically target expression of the eGFP -L10a fusion the eGFPL10a tagged polysome populations . Since specifi protein to defined CNS cell populations in vivo , BAC cally expressed genes are often found in distinct cell types transgenic mice were created (FIG . 4 represents the BAC in physically separable brain structures, several lines offer array strategy ; FIGS. 5 and 6 represent engineering of BAC 30 opportunities for analysis of many cell types not included in vectors and the creation of mice carrying BACs ). To tag this example . Thus , the eGFP -L10a fusion protein is abun mRNAs in specific cell types of the mouse, cell specific cis dant in hippocampal CA1 cells in the Cck BACarray line , and trans regulatory elements and known transcriptional allowing translational profiling ofmRNAs expressed in this units were utilized as described herein and in (Gong et al . cell type ( panel 25 ) . Creation of a BACarray anatomic Nature Vol 425 , 2003 ) . Mouse lines with specific expression 35 database allows the user to browse through serial brain in different subsets of cells in the cerebral cortex ( FIG . 7 ) or sections for each of the lines presented here to determine hypothalamus ( FIG . 8 ) from BAC array lines are presented whether a cell type of interest can be analyzed in one of the by way of example . Detailed anatomic characterization of BACarray lines presented in this example . selected BACarray transgenic mouse lines is presented The figure also shows that projection neurons in the below . 40 cerebrum can be identified by their pyramidal shape , and 4 ) Anatomic Characterization of BACarray Transgenic broadly classified by their laminar specificity, dendritic Mouse Lines arbor, and axonal targets. Lines which clearly label the large Detailed anatomic studies were conducted for BACarray pyramidal cells of layers 6 (Ntsr1 , panel 16 ) , 5b (Glt25d2 , transgenic mouse lines as displayed in FIG . 9 . For each line , panel 17 ) , and 5a ( Etv1 , panel 18 ) have also been repro transgene expression was assayed by immunohistochemistry 45 duced . Morphometric studies provide additional data indi (IHC ) using an antibody against eGFP . IHC data from serial cating that the GENSAT GFP lines and eGFP - L10a BAC coronal sections of the whole brain were collected , scanned array lines target similar cortical pyramidal cell populations at high resolution and mounted for inspection . All sections FIG . 10 shows that BACarray cortical pyramidal cells have were processed equivalently (Neuroscience Associates ) so the same laminar distribution as eGFP GENSTAT lines. The that apparent differences in staining intensity accurately 50 figure shows: A ) Graph (mean + / - SE ) of the distance of reflect differences in transgene expression level. The figure GFP + cell somas from the pial surface for the BACarray shows thumbnail images for each of the 24 cell populations and GENSAT EGFP lines for each pyramidal cell BAC selected for microarray analysis , and high resolution data to driver. The depth of the cells was consistent between both illustrate the morphology evident from IHC analysis of lines for each driver . B ) The percentage of cells in 100 eGFPL10a expression . The regions covered in this charac - 55 micron bins is shown as a histogram of the distribution of terization include the cerebellum (panels 1 - 9 ) , the spinal cell depths for each line in A . The BACarray line and eGFP cord (10 ), the basal forebrain and corpus striatum ( 11 - 14 ), line for each driver had overlapping distributions of cell the brainstem ( 15 ), and cerebral cortex ( 16 -25 ). depths . For well characterized cell types , anatomic confirmation FIG . 11 shows that in many BACarray lines , the presumed of the cells targeted for BACarray analysis was facilitated . 60 cellular identity of the targeted lines was confirmed using For example , given the well described cytoarchitecture of double immunofluorescence for the eGFP -L10a fusion pro the cerebellum , BACarray lines for Purkinje cells (Pep2 , tein and well characterized cell type specific markers. In panel 1 ) , granule cells (Neurodl , panel 2 ) , Golgi neurons some cases, the cell type specific markers corresponded with (Grm2 , panel 3 ) , unipolar brush cells (Grp , panel 5 ) , Berg - the BAC drivers chosen for modification ( Olig2 , Aldh111 , mann glia (Sept4 , panel 8 ), and astrocytes ( Aldh111 , panel 65 Grm2, Chat) . In other cases, commonly used markers which 9 ) were easily identified from the IHC data presented . have been well characterized for specific cell types were Purkinje cells can be recognized in the eGFP -L10a IHC data used , as seen in panel B . In most cases , these studies US 9 ,816 ,096 B2 43 44 established that BAC drivers limited expression to well - subsequent generations were bred to either Swiss - Webster or defined cell populations. There were also several BACarray c57b1/ 6 wildtype mice. Lines were maintained as trans lines in which the transgene is expressed in two or more cell heterozygotes . types. For example , the immunofluorescence ( IF ) analysis of the Lypd6 BA?array line revealed that eGFP -L10a is found 5 TABLE 4 in all Pvalb positive and NeuN negative interneurons of the cerebellar molecular layer, suggesting that this line is valu List of BACs utilized for transgenesis able for analyses of both stellate and basket cells . Finally , in Included are the list of abbreviations used throughout for certain lines it is apparent that the eGFP -L10a transgene is each gene, and the ID for the BAC clone that was modified . expressed in a only a subset of a particular cell type . For 10 Abbreviation Full Gene Name BAC instance , as seen in panel B , in the Grp BACarray line the Sept4 Septin 4 RP23 - 21 N23 eGFP -L10a fusion protein is restricted to the subpopulation Aldhili Aldehyde Dehydrogenase 1 family , RP23 -7N19 of unipolar brush cells (Nunzi et al. , 2002 ) which are member L1 Cmbm5 CKLF- like MARVEL transmembrane RP24 - 317F19 immunoreactive for Grml but not Calb2 ( calretinin ) . domain containing 5 BACarray lines whose expression did not conform to 15 Cck Cholecystokinin RP23 - 234117 readily identified cell types , were also analyzed by IF Chat Choline Acetyltransferase RP23 - 431D9 analysis to provide data concerning the broad classification C ort Cortistatin RP23 - 281A14 of cell populations targeted . For example , in the cerebral Drd2 Dopamine receptor 2 RP23 - 161H15 Drd1 Dopamine receptor DIA RP23 - 47M2 cortex of the Cort BACarray line , Calbl was detected in Grp Gastrin - Releasing Peptide RP23 - 179M10 nearly 50 % of eGFP -L10a positive cells, Pvalb was found in 20 Grm 2 Glutamate Receptor, Metabotropic 2 RP23 - 335E12 less than 5 % of these cells , and Calb2 was not detected . Glt25d2 Glycosyltransferase 25 Domain RP23 - 160M1 Characterization of the fusion protein in the cortex of Pnoc containing 2 BACarray mice revealed that the majority of eGFP -L10a Lypd6 LY6 /PLAUR domain containing 6 RP23 - 14024 NeuroD1 Neurogenic Differentiation 1 RP24 - 151C22 positive cells in the superficial layers of the cerebral cortex Ntsr1 Neurotensin Receptor 1 RP23 - 314D14 are multipolar and are GABA positive , although some cells 25 Olig2 Oligodendrocyte Transcription Factor 2 RP23 - 356P18 in deeper layers of cortex are GABA negative and appear to Pnoc Prepronociceptin RP23 - 264L8 have a single apical dendrite . The multipolar cells in this Pcp2 Purkinje Cell Protein - 2 RP24 - 186D18 case are often positive for Calb2, but not Calbl or Pvalb . Etv1 Ets1 Variant Gene 1 RP23 - 250K4 Both IHC and IF studies of the cortex of the Cck BACarray line clearly demonstrate that eGFP -L10a is detected in small 30 Immunohistochemistry : Six to twelve week old mice were neurons positive for Calb1 but not Pvalb or Calb2 , as well euthanized with CO2 and perfused transcardially with phos as in pyramidal cells ( data not shown ) , consistent with phate buffered saline ( PBS ) PH 7 . 4 followed by 4 % para previous ISH data (found on : the world wide web at stju - formaldehyde in PBS . For diaminobenzidine tetrahydro degem . org ; world wide web at brain -map .org ) ( Lein et al ., chloride ( DAB ) immunohistochemistry, fixed brains were 2007 ; Magdaleno et al ., 2006 ) . 35 treated overnight with 20 % glycerol and 2 % dimethylsul In addition to neuronal cell types, three BACarray lines foxide to prevent freeze - artifacts. Multiple brains ( up to 25 for glial cell types were generated which were analyzed in per block ) were embedded in a gelatin matrix using Multi both cerebellar and cortical tissue. These glial cell types BrainTM Technology (NeuroScience Associates , Knoxville , included astrocytes, mature oligodendrocytes , and a mixed Tenn . ). After curing , the block was rapidly frozen to - 70° C . oligodendroglial line that included mature oligodendrocytes 40 by immersion in a mixture of isopentane and crushed dry and oligodendrocyte progenitors (also called synantocytes ice, and mounted on a freezing stage of an AO 860 sliding or polydendrocytes) (Butt et al. , 2005 ) . Astrocytes were microtome. The MultiBrainTM block was sectioned coro targeted using a BAC for the gene Aldhill that has previously nally at 40 microns . All sections were collected sequentially been described as astrocyte specific (Anthony and Heintz , into the wells of a 4x6 plate filled with Antigen Preserve 2007 ; Cahoy et al. , 2008 ) . This BAC drove transgene 45 solution (50 % PBS pH 7 . 0 , 50 % Ethylene glycol, 1 % expression in both Gfap + ( reactive ) and Gfap , astrocytes, as Polyvinyl Pyrrolidone ) . well as Bergmann glia . It did not express in Ng2 + oligo - After blocking with hydrogen peroxide and serum , the dendrocyte progenitors , nor in Cnp + myelinating oligoden - sections were incubated with a 1 : 75 ,000 solution of Goat drocytes . In contrast, a BAC for the Olig2 transcription anti - eGFP serum (Heiman et al) overnight at room tempera factor directed expression specifically into both the Ng2 + 50 ture . Following extensive washing , the sections were incu and Cnp + oligodendrocyte lineage cells . Finally , a BA?ar - bated with biotinylated secondary antibody ( Anti Goat IgG , ray line for Cmtm5 expressed specifically , albeit weakly , in Vector Labs , Burlingame , Calif .) , washed again , and incu mature (Cnp + ) oligodendrocytes . With these three lines , the bated with avidin -biotin -HRP (Vectastain elite ABC kit , translational profile of the three major classes of glia across Vector Labs , Burlingame, Ca ) according to the manufactur the CNS can be examined . 55 er ' s instructions . Sections were again washed and incubated In cases of relatively weakly expressing lines , such as with DAB and hydrogen peroxide until fully developed . Cmtm5, new drivers can be selected to more effectively Finally , developed sections were mounted on gelatinized target the same cell type . Studies with a mature oligoden ( subbed ) glass slides , air dried , dehydrated in alcohols , drocyte line (Cnp JD368) have demonstrated improved cleared in xylene and coverslipped . Images were acquired RNA yield and data quality from a more strongly expressing 60 with a Zeiss Axioskop2 microscope with a 10x ( 1 . 5 NA ) transgene (FIG . 12 ). objective using an automated x , y stage (Marzhauser scan8 ) 5 ) Methods controlled by a PC with Zeiss KS400 software running BAC Modification , Transgenesis , and Animal Husbandry : custom macros. Wildtype brains showed no labeling . BACs from For fluorescence immunohistochemistry , fixed brains Table 4 were modified as described to insert an eGFP - 65 were cryoprotected in 30 % sucrose in phosphate buffered L10a fusion protein into the translation start site of the driver saline ( PBS ) , frozen , cut to 40 micron serial floating sections gene (Gong et al. , 2002 ; Gong et al. , 2003 ) . Founders and by cryostat, and stored in PBS 0 . 1 % Sodium Azide at 4° C . US 9 ,816 , 096 B2 45 46 until use . Sections were blocked in PBS with 5 % normal rogen Corporation , Carlsbad , Calif. ) for 1 h at room tem donkey serum , 0 .25 % triton for 30 minutes, then incubated perature. Sections were washed with PBS / 0 .05 % Tween - 20 overnight with primary antibodies ( Table 5 ) . Sections were and a fluorescent HRP substrate , Tyramide- AlexaFluor 546 washed in PBS , exposed to appropriate Alexa dye conju conjugate ( from TSA kit # 13 , Invitrogen Corporation , Carls gated secondary antibodies (Molecular Probes/ Invitrogen , 5 bad , Calif. ) , was deposited on the slides. Slides were washed Carlsbad , Calif. ) for 90 minutes , washed , then mounted . All again with PBS/ 0 .05 % Tween - 20 , mounted with Prolong images were acquired with a Zeiss Inverted LSM 510 laser Gold Antifade ( Invitrogen Corporation , Carlsbad , Calif. ) , scanning confocal microscope , with a Z thickness of 2 dried overnight, and fluorescence was visualized on a Zeiss microns. Z - stacks through the sections were acquired to LSM510 confocal microscope (Carl Zeiss , Thornwood , confirm colocalization . N . Y .) . TABLE 5 Antibodies, product numbers , and sources used for immunohistochemistrystry .. Symbol Antibody Name Product Number Source EGFP Goat Anti - EGFP NA (Heiman et al. ) EGFP Chicken Anti - EGFP AB19370 Abeam , Cambridge Ma. NEUN Neuronal Nuclei MAB377 Chemicon , Temecula , Ca. OLIG2 Oligodendrocyte transcription factor 2 AB9610 Chemicon , Temecula , Ca. CSPG4 Ng2 Proteoglycan AB53420 Chemicon , Temecula , Ca . CHAT Choline Acetyl- Transferase AB143 Chemicon , Temecula, Ca . GRM2 /GRM3 metabotropic glutamate receptor 2 & 3 AB1553 Chemicon , Temecula , Ca . GRM1 metabotropic glutamate receptor 1 AB1551 Chemicon , Temecula , Ca . CNP1 Cnpase MAB326R Chemicon , Temecula , Ca . CALB1 Calbindin 300 Swant, Bellinzona , CH CALB2 Calretinin 6B3 Swant, Bellinzona, CH PVALB Parvalbumin Pv28 Swant, Bellinzona , CH GFAP Glial Fibrillary acidic protein Z0334 Dako , Denmark S100 S100 Z0311 Dako , Denmark GLUL Glutamine Synthetase G2781 Sigma- Aldrich , St Louis , Mo . SLC18a3 Vesicular Acetylcholine Transporter SC -7717 Santa Cruz Biotech , Santa Cruz, Ca . ALDH1L1 10 - Formyltetrahydrofolate NA a gift from Dr. Robert Cook Dehydrogenase GABA A2052 Sigma- Aldrich , St Louis , Mo.

Fixed Sections: For fixed sections, BACarray transgenic Fresh Frozen Sections : For all nuclear immunohistochem mice were deeply anesthetized with pentobarbital or 50 35 istry (Anti - Atrx , Anti -PML , Anti -yH2A . X ) , fresh frozen mg/ ml Nembutal and transcardially perfused with 10 ml of brain sections were used . For fresh frozen brain sections , phosphate buffered saline (PBS ) followed by 40 ml of 4 % BACarray mice were euthanized with CO2, decapitated , and their brains removed into ice - cold PBS . Brains were then paraformaldehyde ( PFA ) in PBS . Brains were dissected and embedded in Neg50 , placed on dry ice for an hour, and post - fixed at room temperature for exactly 1 h with 4 % PFAORS 40 stored until needed at - 80° C . Before sectioning, frozen in PBS . After fixation , brains were washed 3 times in PBS 40 blocks of tissue were equilibrated at - 20° C . , and 16 um and incubated in 5 % weight/ volume ( w /v ) sucrose in PBS at sections were cut on a Leica cryostat. Slides with fresh 4° C . for 1 hour with gentle agitation . Brains were then frozen brain sections were stored at - 80° C . or - 20° C . until incubated for 24 h in 15 % w / v sucrose in PBS at 4° C . with further use . gentle agitation and for 24 h in 30 % w /v sucrose in PBS alat 45 Prior to use, sections were allowed to air - dry at room 4° C . with gentle agitation . Brains were placed in an temperature for 15 minutes , then fixed with 1 % freshly made embedding mold filled with Neg -50 embedding medium PFA in 1xPBS for 10 minutes at room temperature . Next, (Richard Allan Scientific , Kalamazoo , Mich .) for 1 hour at slides were washed three times with Ix PBS , and permea room temperature , and were subsequently incubated on dry bilized for 10 minutes at room temperature with 0 . 05 % ice for 1 hour to freeze the embedding medium . Brains were 50 Triton X - 100 in PBS . After permeabilization , tissue sections then transferred to and stored at - 80° C . until sectioned . 12 were blocked using either 5 % horse serum or 5 % goat serum um sagittal sections were cut, mounted on glass slides, and in PBS for at least one hour. Sections were incubated with kept overnight at - 20° C ., then transferred to - 80° C . until primary antibodies overnight in a humidified chamber at 4° used for immunohistochemistry . C . , washed three times with PBS , and incubated at room Before use sections were thawed and dried at room 55 temperature for one hour with appropriate secondary anti temperature for 20 min , washed with PBS , and incubated in bodies . After incubation with secondary antibodies , sections 0 . 2 % H202 /PBS at room temperature for 30 min to quench were incubated for 10 minutes with the nuclear stain TO endogenous peroxidase activity . Sections were washed with PRO - 3 ( Invitrogen Corp ., Carlsbad , Calif .) , washed three PBS , permeabilized with PBS / 0 .05 % Tween - 20 , and times with PBS , and coverslipped with Aquamount mount blocked with Image - it FX signal enhancer (Invitrogen Cor - 60 ing medium ( Lerner Laboratories , Pittsburgh , Pa . ) . Images poration , Carlsbad , Calif . ) for 30 min at room temperature . were collected on an upright Zeiss Axioplan LSM 510 laser Sections were washed with PBS / 0 .05 % Tween - 20 and scanning confocal microscope ( Carl Zeiss MicroImaging , blocked again with 2 % donkey serum / 0 . 1 % fish gelatin / Inc. , Thornwood , N . Y .) using 40x / 1 . 2 , water immersion PBS/ 0 . 05 % Tween - 20 . Sections were then incubated over objective , 63x / 1 . 4 oil immersion , and 100x / 1 . 4 oil immer night at 4° C . with primary antibodies . The next day , sections 65 sion lenses . were washed with PBS / 0 .05 % Tween - 20 and incubated with Quantification of Laminar Position of Cortical Pyramidal anti- rabbit Superpicture HRP Polymer Detection Kit ( Invit - Cells : Anti - eGFP immunohistochemistry with DAB was US 9 ,816 ,096 B2 47 48 performed on 20 micron sagittal sections from the Etv1 Mountain View , Ca) in 5xSSC , 2 .5xDenhardt ' s solution , TS88 , Glt25d2 DU9, and Ntsr1 TS16 BACarray lines as with 500 ug /ml sheared denatured salmon sperm for 30 described (Gong et al) , and images were acquired as above . minutes , then hybridized overnight with labeled riboprobe . Corresponding digital images of the adult sagittal sections Slides were washed for 5 minutes at 65 C with 5XSSC , then were downloaded from gensat. org for lines expressing eGFP 5 for 1 hour with 0 .2xSSC at 68° C . , and for 5 minutes at room from the same BACs . Sections containing motor cortex temperature . Slides were washed in TBS ( 100 mM Tris HC1, ( corresponding to Paxinos section 111 ) ( Paxinos and Frank - 150 nM NaC1, pH 7 . 5 ) twice for five minutes, blocked with lin , 2001 ) were imported into ImageJ (rsb .info .nih . gov / ij ) . 10 % Roche blocking solution in TBS for one hour , incu The distance from the apical tip of the soma to the pial bated with primary antibodies for 1 hour in the same solution surface was measured using the ' straight line selection ' tool. 10 ( Sheep anti- Dig , alkaline phosphatase conjugated , Roche 11 The apical tip was defined as the site at which the apical 093 274 910 ) and Goat anti - eGFP where appropriate , dendrite and the cell body converge . Only cells with a washed with TBS , and developed with NBT/ BCIP or HNPP / clearly visible apical dendrite and a uniformly stained soma Fast Red following manufacturers protocols (Roche ) . For were measured . At least 50 cells were measured from each eGFP /ISH double fluorescence , sections were then incu image . All measurements were then converted from pixels to 15 bated with alexa - 488 donkey anti- goat antibody ( Invitrogen ) microns using the following scale : 1 pixel = 1 . 33 microns . for one hour before counterstaining with DAPI, washing and In Situ Hybridization : IMAGE consortium clones con - coverslipping . Images were acquired with Zeiss Inverted taining sequences from genes of interest were purchased LSM 510 laser scanning confocal microscope . from Open Biosystems ( Table 6 ) . Probes were synthesized by linearization of plasmid with appropriate restriction 20 Example 4 enzyme , template purification with Qiagen PCR purification kit, followed by in vitro transcription with appropriate Isolation and Purification of Ribosome- mRNA enzyme ( T3 or T7 ) using DIG RNA labeling kit (Roche , Complexes In Vivo Basel, CH ). Labeled RNA was purified with ProbeQuant G -50 microcolumns (GE Healthcare ), and assayed for qual- 25 Improved procedures for rapid extraction and immunoaf ity and quantity with a Bioanalyzer, following manufactur finity purification of the eGFP - tagged functional polyribo er' s instructions (Agilent Technologies, Santa Clara Calif. ) . some complexes from in vivo intact brain tissue were TABLE 6 List of plasmids of in situ hybridization studies: ISH was conducted on 11 genes enriched in either Grm2 positive granule cell layer interneurons, or Pnoc positive neurons of cortex , using plasmids ordered from Open Biosystems. Plasmids were sequenced to confirm the accuracy and orientation of the ESTs . Plasmids were linearized with either Sal I or Eco RI and Dig labeled RNA was transcribed with either T3 or 17 polymerase to create anti - sense RNA probe . Genbank Open Biosystems # Gene Name Enzymes PNOC Gene ID B230118H07Rik BC025075 MMM1013 - 7513748 RIKEN cDNA B230118H07 gene Sal I cut, T7 polymerase Chd4 BC058578 MMM1013 - 9201124 chromodomain helicase DNA Sal I cut, T7 polymerase binding protein 4 Cidea AA061879 EMM1002 - 1115733 cell death - inducing DNA Eco RI cut, T3 polymerase fragmentation factor, alpha subunit- like effector A Crabp1 BC065787 MMM1013 - 9202137 cellular retinoic acid binding Eco RI cut , T3 polymerase protein I Ddefi BC094581 MMM1013- 98479313 development and differentiation Eco RI cut, T3 polymerase enhancing Igf1 BC012409 MMM1013 - 65370 insulin - like growth factor 1 Sal I cut , T7 polymerase GRM2 Gene ID Lypd1 CA328245 EMM1002- 6960067 RIKEN DNA 2700050C12 gene Eco RI cut, T3 polymerase Slc6a5, GlyT2 BM941867 EMM1032 - 584726 solute carrier family 6 Eco RI cut , T3 polymerase (neurotransmitter transporter , glycine ) , member 5 Amn AA023455 EMM1002- 1077429 amnionless Eco RI cut, T3 polymerase Penki AA098193 EMM1002- 1183311 preproenkephalin 1 Eco RI cut , T7 polymerase Gcdr BC031885 MMM1013 -7511930 glucagon receptor Sal I cut, T7 polymerase Ceacam 10 BC003346 MMM1013 - 62963 CEA -related cell adhesion Sal I cut, T7 polymerase molecule 10

Adult mice were processed as above , and brains were cut 60 developed and optimized ( A . Alexa, J. Rahnenfuhrer, T . on cryostat into 20 micron sections and mounted onto Fisher Lengauer, Bioinformatics 22 , 1600 - 7 (2006 ) ). Highly puri Superfrost Plus slides . Tissue was washed , then post fixed fied RNA and protein was consistently obtained from BAC for 20 minutes in 4 % paraformaldehyde PBS , permeabilized array mice . Key steps of the purification protocol included with 0 .05 % Triton X100 , digested with 50 ug /ml Proteinase rapid manual dissection and homogenization of the tissue in K in TE for 10 minutes, and acetylated for 10 minutes in 65 question , inclusion of magnesium and cycloheximide in the 0 . 1M TEA with 0 . 25 % acetic anhydride . Sections were lysis buffer to maintain ribosomal subunits on mRNA during prehybridized in Atlas hybridization chambers ( Clontech , purification , inhibition of endogenous RNase activity , solu US 9 ,816 ,096 B2 49 50 bilization of rough endoplasmic reticulum -bound polysomes Example 5 under nondenaturing conditions, use of high - affinity anti eGFP antibodies, and the addition of high -salt washes after Analysis of mRNA from BA?array Mice immunoaffinity purification to reduce background . FIG . 13 displays purification of eGFP -tagged L10a and co -purifica - 5 1 ) Preparation of mRNA tion of untagged ribosomal protein L7 from D1 BACarray For mRNA purification , mice were decapitated and the animals but not wild - type littermates in ( a ) (D1 , samples specific tissue of interest was quickly dissected from mice brains. Pooled tissue was immediately collected in ice - cold from D1 BACarray mice; WT, samples from wild - type dissection buffer and homogenized in ice - cold polysome littermates ; In , 1 % Input; UB , 1 % Unbound ; IP , 6 . 5 % 10 extraction buffer ( 10 mM HEPES (pH 7 . 4 ] , 150 mM KC1, 5 Immunoaffinity purified sample ). eGFP -L10a signal is only mM MgCl2 , 0 . 5 mM dithiothreitol, 100 ug /ml cyclohexim present in the D1 IP lane because the IP samples were more ide , protease inhibitors , and recombinant RNase inhibitors ) concentrated relative to In and UB . In ( b ) purification of 18S using a motor -driven Teflon - glass homogenizer. Homoge and 28S rRNA from D1 BACarray transgenic animals ( top nates were centrifuged for 10 minutes at 2 ,000xg , 4° C ., to panel) but not wild type littermates ( bottom panel) ) . asas 15 pellet large cell debris , and NP- 40 or IGEPAL (EMD Bio detected by Bioanalyzer PicoChips ( Agilent Technologies ) sciences, San Diego , Calif. ; Sigma, St. Louis , Mo . ) and is shown . 28S rRNA runs at ~ 47 sec , 18S rRNA runs at ~ 43 DHPC ( Avanti Polar Lipids, Alabaster , Ala .) were added to sec, and the marker peak runs at - 23 sec . the supernatant at a final concentration of 1 % and 30 mM , Following immunoprecipitation of ribosomal complexes , respectively . After incubation on ice for 5 minutes, the electron microscopy was utilized to visualize the ribosomes . 20 clarified lysate is centrifuged for 10 minutes at 13 ,000xg to Aliquots of anti -GEPGFP coated magnetic beadsheads were fixed inin pellet unsolubilized material. Goat anti -GFP ( custom made ) 2 . 5 % glutaraldehyde /0 . 1M cacodylate (pH 7 .41 on ice . The coated protein G Dynalmagnetic beads ( Invitrogen Corpo ration , Carlsbad , Calif .) are added to the supernatant and the bead pellet was post - fixed with 1 % osmium tetroxide in the mixture was incubated at 4° C . with end - over - end rotation same buffer on ice . After treatment with 0 . 5 % aqueous 25 for 30 minutes . Beads were subsequently collected on a uranyl acetate at room temperature , the specimen was dehy magnetic rack , washed three times with high - salt polysome drated with graded alcohol ( 70 , 90 , 100 % ) and treated with wash buffer ( 10 mM HEPES [ pH 7 . 4 ) , 350 mM KC1, 5 mM propylene oxide before embedding in Embed 812 resin . The MgCl2 , 1 % NP - 40 , 0 .5 mM dithiothreitol, 100 ug/ ml cyclo resin was polymerized in a 60° C . oven for 2 - 3 days. Silver heximide ) and immediately placed in Tri Zol- LS reagent sections were cut with a Dupont diamond knife on aa Reich -- 30 ans( Invitrogen Corporation , Carlsbad Calif .) and chloroform to ert - Jung UltraCut E ultramicrotome. The sections were extract the bound rRNA and mRNA from polysomes . After collected on copper grids, doubly stained with saturated , extraction , RNA was precipitated with sodium acetate and aqueous uranyl acetate and lead citrate before examination Glycoblue (Ambion , Austin , Tex .) in isopropanol overnight with a Jeol 100cx electron microscope ( JEOL , Peabody, at - 80° C ., washed twice with 70 % ethanol, resuspended in Mass. ) operated at 80 kV (FIG . 14 ) . 35 water , and further purified using an Rneasy Micro Kit 1) Generation of Monoclonal Antibodies ( Qiagen , Valencia , Calif. ) with in - column DNase digestion . All immunoprecipitations except for the Drdl and Drd2 Purified samples were analyzed using a Bioanalyzer (Agi lines , which used the Goat Anti - eGFP described in later lent Technologies, Santa Clara , Calif .) in order to assess examples, were done using two monoclonal anti - eGFP 40 mRNAintegrity quantity . and quality , as reflected by rRNA levels and antibodies ( clones 19C8 and 19F7 ) specifically generated for After purification of RNA samples, 1 . 5 ul was used for this purpose at the Monoclonal Antibody Core Facility at quantification using a Nanodrop spectrophotometer (Nano Memorial Sloan -Kettering cancer center. Mice were immu - Drop Technologies, Wilmington , Del. ) . Each sample was nized with purified GST - eGFP fusion protein and several further analyzed with a Bioanalyzer (Agilent Technologies , rounds of screening were performed to identify clones which 45 Santa Clara , Calif .) to ensure that the quality of each RNA functioned well in immunoprecipitation assays. Initially , sample met standard criteria and that they were comparable monoclonal supernatants were tested by ELISA using 96 to each other . Specifically , all RNA samples had a 260 / 280 well plates coated with eGFP purified from transiently ratio (Nanodrop ) of at least 1 . 8 , and a RNA Integrity transfected 293T cells . Next, positive clones were screened Number (Bioanalyzer ) of at least 7 . Moreover , the quality of in immunoprecipitation assays , again using the eGFP puri- 50 the samples was visually estimated using the Bioanalyzer fied from transfected 293T cells . Finally , positive clones readout to estimate the level of potential degradation or which strongly immunoprecipitated eGFP from lysates from contamination for each sample . 2 ) Microarray Analysis of mRNA a transgenic mouse line expressing eGFP under the BAC Once these criteria were met, a total of 15 ng of RNA from driver of interest were identified . 55 each sample was amplified , biotinylated , and fragmented 2 ) Immunoprecipitation of Polyribosomes with the Affymetrix two -cycle amplification kit (Affymetrix , Immunoprecipitation of polyribosomes and isolation of Santa Clara , Calif. ). After amplification , samples were again mRNA was done as described in detail below , except goat quantified with a Nanodrop spectrophotometer, and 20 ug of anti -eGFP antibody was substituted with a mix of two amplified RNA was fragmented following the Affymetrix monoclonal eGFP antibodies ( 19C8, 19F7) . Three to six 60 protocol. Amplified and fragmented samples were analyzed mice for each replicated sample were euthanized with CO2 with a Bioanalyzer before hybridization to Affymetrix and distinct brain regions ( cerebellum , cortex , corpus stria - mouse 430 2 .0 microarrays . All hybridizations were done tum , basal forebrain , brainstem , or spinal cord ) were dis according to standard Affymetrix protocols at the Rock sected Each cell population was assayed in triplicate . RNA efeller University Genome Array Center. quality control, amplification and hybridization were done 65 Tissue handling and RNA purification for all samples was as described by Heiman et al. For consistency across cell as described . Purified RNA was converted to double types, 15 ngs of total RNA were amplified for each sample . stranded cDNA using the SuperScript GeneChip Expression US 9 ,816 ,096 B2 51 52 3 ' Amplification Reagents Two - Cycle cDNA Synthesis Kit ( < 50 ) from analysis , as well as those probesets identified as (Affymetrix , Santa Clara , Calif .) and the GeneChip monoclonal background ( Table 7 ) , and replicate samples T7 -Oligo ( dT ) Primer (Affymetrix , Santa Clara , Calif. ). were averaged . cDNA was used for the in vitro synthesis of RNA using the MEGAscriptT7 Kit ( Ambion , Austin , Tex . ). CRNA was 5 TABLE 7 purified using the GeneChip Sample Cleanup Module ( Af List of Probesets excluded from analysis fymetrix , Santa Clara, Calif. ). 600 ng or less of clean CRNA To identify mRNAs which interact with monoclonal antibodies or was used in the second - cycle cDNA synthesis reaction using protein G dynabeads in the absence of eGFP , BACarray was the SuperScript GeneChip Expression 3 '- Amplification performed on a wildtype mouse brain and compared to unbound whole brain RNA . The excluded probesets listed here are those Reagents Two -Cycle cDNA Synthesis Kit (Affymetrix , for genes found to be highly enriched ( > 6 fold ) in the wildtype Santa Clara , Calif . ) and random primers ( Affymetrix , Santa IP /UB as well as enriched in multiple IPs from diverse regions Clara , Calif .) . The cDNA was purified using the GeneChip and cell types . Sample Cleanup Module ( Affymetrix , Santa Clara , Calif . ) . Probeset IP /UB WT Symbol Name Purified cDNA was used for the in vitro synthesis of 15 biotin - labeled cRNA using the GeneChip IVT Labeling Kit 1428909 _ at 374 . 4 1200015M12 Rik novel 1428720 _ s _ at 41. 55 2010309G21Rik novel ( Affymetrix , Santa Clara , Calif. ) . CRNA was purified using 1453238 _ s _ at 57 . 87 3930401B19 Rik novel the GeneChip Sample Cleanup Module ( Affymetrix , Santa 1435640 _ x _ at 159 C8506T novel Clara , Calif. ) and fragmented into 35 - 200 base pair frag 1425247 _ a _ at 37 . 25 Igh - 4 immunoglobulin heavy chain 4 iserum IgG ') ments using a magnesium acetatoacetate bufferun lu ( Affymetrix o ,, wanaSanta 20 14253241474 _ x _ at 11 .34 Igh - 4 immunoglobulin heavy Clara , Calif . ) . chain 4 iserum IgG ' ) As controls, Affymetrix standard spike - in controls ( eu - 1424305 _ at 14 . 27 Igj immunoglobulin joining chain karyotic hybridization kit ) were used . 1452417 _ x _ at 68 . 89 Igk - V28 immunoglobulin kappa For hybridization procedures and parameters , 10 micro chain variable 8 ( VE ) grams of labeled cRNA were hybridized to Affymetrix 25 1427455 _ x _ at 60 . 27 Igk - V28 immunoglobulin kappa GeneChip Mouse Genome 430 2 . 0 arrays (available using chain variable 8 ( VE ) 1427660 _ x _ at 58 .46 Igk - V28 immunoglobulin kappa the hypertext transfer protocol on the world wide web at chain variable 8 (VE ) affymetrix .com /products / arrays/ specific /mouse430 _ 2 . affx ) 1456162 _ x _ at 861. 2 Lmna lamin AC for 16 h at 45° C . The GeneChips were washed and stained 1455892 _ x _ at 6 .874 Lmna lamin AC according to the manufacturer 's recommendations (Affyme - 30 1450009 _ at 12 .58 Ltf lactotransferrin 1456456 __ x _ at 122 . 6 Mela melanoma antigen trix , Santa Clara , Calif. ) using the GeneChips Fluidics 1427797 _ s _ at 42. 54 Mnda myeloid cell nuclear Station 450 ( Affymetrix , Santa Clara , Calif .) . This procedure differentiation antigen included staining the chips with phycoerythrin - streptavidin , 1427798 _ x _ at 313 .97 Mnda myeloid cell nuclear signal amplification by a second staining with biotinylated differentiation antigen 1452556 _ at 14 . 87 Mnda myeloid cell nuclear anti - streptavidin , and a third staining with phycoerythrin - 35 differentiation antigen streptavidin . 1416957 57 .24 Pou2af1 Pou domain , class 2 , For array design , Affymetrix Mouse Genome 430 2 .0 associating factor 1 arrays were used in all experiments . Information regarding 1435697 _ a _ at 9 . 079 Pscdbp pleckstrin homology , Sec7 and coiled - coil the array design and features can be found at the hypertext domains , binding protein transfer protocol on the world wide webweb atdi anymenxaffymetrix . com .. 40 14367271436 _ x _ at23 .84 Sptlci serine For measurement data and specifications , Mouse Genome palmitoyltransferase , 430 2 . 0 arrays were scanned using the GeneChip Scanner long chain base subunit 1 3000 (Affymetrix , Santa Clara , Calif .) . Three biological 1435137 _ s _ at 53 .47 novel replicates were performed for each experiment. GeneChip 1427932 _ s _ at 26 .78 novel CEL files were subjected to Harshlight analysis to detect if 45 any blemishes were present on the GeneChips ( available Each IP was then compared to the unbound samples from using the hypertext transfer protocol at asterion .rockefell - the same tissue to calculate a ratio of IP /UB as a measure of er. edu /Harshlight / index2 .html ) (M . Suarez - Farinas, M . Pel - enrichment' . UB samples generally show little or no deple legrino , K . M . Wittkowski, M . O . Magnasco , BMC Bioin - tion of cell - specific RNA following IP , and UB samples from formatics 6 , 294 (2005 ) ) . Only GeneChips without major 50 several different IPs from the same tissue were averaged for blemishes were used . GeneChip CEL files were imported each comparison . UB samples from corpus striatum ( Chat into Genespring GX 7 . 3 . 1 (Agilent Technologies, Santa line) and neostriatum (Drdl and Drd2 lines ) were normal Clara , Calif . ) , processed with the GC -RMA algorithm , and ized together. IPs were globally normalized to UBs using the expression values on each chip were normalized to that Affymetrix biotinylated spike in controls , to correct for any chip ' s 50th percentile . Data were filtered to eliminate genes 55 broad biases in scanning and hybridization . For each cell with intensities in the lower range . Only genes where more type , Table 9 contains the IP /UB values for all genes with than one sample had a normalized intensity larger than 16 ( 4 fold change > 2 and p < 0 .05 by Welch 's t - Test, with Benja in log2 scale ) were kept in the analysis . Statistical analysis mini and Hochberg FDR multiple testing correction , as to determine which genes are differentially expressed in the calculated by Genespring GX version 7 . 3 (Agilent ) . different conditions was carried out using the Limma pack - 60 For three cell populations, a further ' corrected enrich age from Bioconductor project (available using the hyper ment ' was calculated . For the Bergmann glial line , Sept4 , text transfer protocol on the world wide web at bioconduc - which also shows low level expression in mature oligoden tor. org ) . drocytes , the corrected enrichment is the intersection of the MIAME compliant raw data are available from the IP /UB analysis described above with a comparison of the APNRR server and GEO . Replicate array samples were 65 Bergman Glial line with the mature oligodendrocyte line , normalized with quantile normalization (GCRMA ) . Data Cmtm5, using the same fold change and statistical criteria were filtered to remove those probesets with low signal applied above. For unipolar brush cell line Grp , which also US 9 ,816 ,096 B2 53 54 shows expression in some Bergmann glia , the corrected wide translational profile via microarray , using the translat enrichment is the intersection of the IP /UB enrichment ing ribosome affinity purification ( TRAP ) methodology of described above with a comparison of Grp with the Bergman Heiman et al (2008 ) . For each cell type, pooled microdis glial line , using the same criteria . Finally, a substantial sected tissue from three to six transgenic mice was used . As proportion of the RNA in the cerebellum is generated by 5 shown in FIG . 16 , this procedure yielded the purification of granule cells , and thus the UB samples are highly enriched GFP -ribosomal fusion protein along with cell specific in granule cell RNA . Therefore , to identify granule cell mRNAs. The yield of total RNA from the initial IP is genes, the ' corrected enrichment was calculated by com dependent on the number of labeled cells in the tissue and paring granule cell IP to the average of all other cerebellar the intensity of the transgene expression within each cell , cell type IPs, using the same criteria as above . 10 with RNA recoveries ranging from tens to thousands of Hierarchical clustering was performed in Genespring nanograms per IP . RNA from the unbound (UB ) fraction of using the ' condition tree ' function with a smoothed corre the immunoprecipitation was harvested to measure the genes lation metric on the GCRMA normalized data for the 20 % expressed in the dissected region as a whole . IP and UB of probesets with the highest coefficient of variation . mRNA were then amplified into labeled RNA using stan Shannon entropy was calculated in excel from GCRMA 15 dard protocols . normalized values with the following procedure : After Labeled cRNAs from the IP and UB fractions were then excluding probesets with no signal> 100 in at least one hybridized onto Affymetrix mouse 430 2 . 0 microarrays . sample , normalized expression measurements for each data Samples derived from multiple independent pools of mice set were categorized into 5 bins by log base 10 values ( 1 - 9 , ( replicates ) were assayed for each cell type. FIG . 17 presents 10 - 99, 100 - 999 , 1000 - 9999 , 10000 - 99999 ) , and Shannon 20 these data and illustrates that BACarray data are highly entropy was calculated for each gene across 1 ) just the IP reproducible and cell - type specific samples, 2 ) just the UB samples , and 3 ) across all samples As shown in Panel A , replicates for the same cell type using the following formula (Schneider , 2007 ) . gave nearly identical genome wide translational profiles , confirming the results of Heiman et al, and extending this 25 finding to many other cell types . The average Pearson ' s correlation between replicates for a given cell population H = - EPi log 2Pi from independently isolated samples was above 0 .98 across all cell types . To determine whether the position of integra i = 1 tion of the BACarray construct would influence the data , The 10 % of probesets with the highest and lowest entropy 30 results obtained from independent BACarray founder lines across all samples were analyzed using the BINGO plugin prepared with the same engineered BAC were also exam for the cytoscape software, using the full mouse Gene ined , as presented in D . This analysis revealed that for Ontologies, a p threshold of 0 .01 on hypergeometic tests independent founder lines targeting identical cell popula with Benjamin Hochberg FDR correction for multiple test - tions , the variation in translational profiles between lines ing (Ashburner et al. , 2000 ; Maere et al. , 2005 ) . Results 35 was low , and no more extensive than that seen for replicate from this analysis are substantially similar to those obtained samples isolated from the same BACarray founder line using the EASE online implementation of Gene Ontologies (Panel D ) . Thus, the location of the transgene insertion into and EASE statistic (Dennis et al. , 2003 ) . the genome had little global impact on the data obtained Pearson ' s correlation with MBP was calculated in Gene - from the translating ribosome affinity purification ( TRAP ) spring . 40 methodology. Finally , four differentmonoclonal antibodies Comparative analysis of all cell types , and heat maps were and one goat polyclonal against eGFP were tested . Each generated with the R statistical software. Data were normal antibody immunoprecipitated comparable levels ofmRNA ized as above . Then , for each cell type the total probeset list for the BACarray lines tested ; similar global gene transla was filtered to remove those probesets with signal less than tional profiles were obtained from each antibody tested in 50 , or IP /UB values less than the average plus two standard 45 IPs from specific BACarray lines . A small number of probe deviations of the IP /UB values of the relevant negative sets were consistently enriched in every BACarray dataset control genes, or 1 , whichever was lesser. Then , with this analyzed . Since these same probesets are also enriched in filtered list of genes , a fold change was calculated for this immunoprecipitates from control mice with no transgene cell type versus all other samples , iteratively . For each expression , it was concluded that they represent background comparison , the fold changes were ranked from highest to 50 which have systematically eliminated from further analysis . lowest , and these ranks were averaged across comparisons The enrichment for each mRNA immunoprecipitated for a cell type . The top one hundred probesets from this from the targeted cell type ( IP ) versus its expression in the average ranking were selected for further analysis . tissue sample dissected for the analysis (UB ) was measured . To assess if the ribosomal immunoprecipitation is biased The ratio of IP /UB was calculated , thereby identifying those towards longer transcripts, the signal intensity versus tran - 55 genes which are highly enriched in each cell type. Panel B script length for all probesets was plotted . No positive shows scatter plots for three representative cell types of the correlation between signal intensity and length was detected cerebellum . Differences are evident between the genome for any sample (FIG . 15 ) . wide translational profiles of IP samples compared to whole tissue (UB ), with each cell population displaying a unique Example 6 60 profile of thousands of specifically enriched genes on the microarray . As in Panel C , Venn diagrams constructed from BACarray Polysome Purification , RNA Extraction the top 1000 most enriched probesets for each cell type can and Control Microarray Experiments be used to illustrate this point. Thus, approximately 75 % of these enriched probesets are not shared between cerebellar 1 ) Overview 65 Purkinje cells , granule cells and unipolar brush cells , and In total, twenty four cell populations in five regions were only 52 of the probesets enriched in these three cell types chosen to assay with immunoprecipitation ( IP ) and genome versus whole cerebellum are shared between them . Further US 9 ,816 ,096 B2 55 56 more , the primary data collected from these experiments has result for Ceacam10 expression in granule layer interneu been deposited in the Gene Expression Omnibus (Edgar et rons is not evident in either of the ISH databases (available al ., 2002 ) . on the world wideweb at stjudebgem . org and the world wide FIG . 18 presents the accuracy of this methodology to web at brain -map .org ) , in both cases one can see scattered enrich for cell -specific genes. The BACarray data for known signal in this area that may indicate expression of this cell - specific markers (positive controls ) for each cell type mRNA in cerebellar Golgi neurons . and the BACarray data for genes known to be expressed In order to further validate the BACarray datasets , the exclusively in other cell types (negative controls ) were enrichment of a variety ofmRNAs isolated from the Chat B for 10 (motor neuron ) and Pcp2 (Purkinje cell ) BACarray trans examined . Panel A shows a scatter plot of IP vs. UB for genic lines were measured with quantitative real time PCR spinal cord motor neurons. Probesets for known markers of (QRT - PCR ) (Panel D ) . For all of the control genes tested , this motor neurons with measurable signal on the array are methodology confirmed the BACarray results . For genes not clearly enriched in the IP sample , whereas probesets for glial 15 previously known to be expressed in a specific cell type , cell- specific RNAs, that should not be present in these cells , 13 results from qRTPCR demonstrated that seven out of the are enriched in the UB sample . To establish the generality of eight mRNAs assayed were in fact cell type enriched (Panel this finding, the enrichment in the IP or UB sample was D ). Moreover , despite a negative ISH result , qRT- PCR quantified by calculating an average ratio of IP /UB for validated the expression of Ceacam10 in the cerebellum and positive and negative controls for each cell type where at 20 itsits enrichment in Golgi cells (Panel D ) . In some cases , least three positive controls could be found in the literature. therefore, the translating ribosome affinity purification As shown in Panel B , all IPs showed a clear enrichment for ( TRAP ) methodology appears to be more sensitive than appropriate known markers , (Panel B , plotted in log base 2 ) . ISH . Even for cell types with only one known marker ( Pnoc 25 2 ) Quantitative PCR (qPCR ) of Purified mRNA positive interneurons , and Grp expressing unipolar brush In some experiments , 20 ng of purified RNA was used to cells ), probesets for these genes were consistently and produce cDNA with a NuGEN WT Ovation kit (NuGEN highly enriched in the IP . In the IPs with the lowest relative Technologies, San Carlos, Calif .) and the resulting cDNA yield of RNA , such as those for mature oligodendrocytes 30 was purified and quantified . 10 ng of cDNA was used for ( Panel B ) , and Cort expressing interneurons, background each real- time gene expression assay . Applied Biosystems was proportionally higher , and enrichment was less robust . (Foster City , Calif .) TaqMan pre- designed gene expression Novel cell -specific markers for rare cell types using the assays were used , following the manufacturer' s instructions BACarray approach were identified . Eleven genes predicted 35 and using an Applied Biosystems 7900 Sequence Detection by BACarray to be enriched in either the Grm2 expressing System . interneurons of the granule cell layer (Golgi cells ), or the Alternatively , in some experiments , cDNA was synthe Pnoc expressing cells of the cerebral cortex were screened . sized from 20 ng of total RNA from the three replicate IP and Using confocal microscopy, double immunofluorescence for UB samples using M -MulV reverse transcriptase (M0253L ), both eGFP -L10a fusion protein and the ISH probes were from New England Biolabs ( Ipswich , Ma) , using oligo evaluated . For the nine genes where ISH gave clear results , dT23VN (SEQ ID NO : 1) as a primer, then purified with the all were clearly overlapping with eGFP -L10a . Qiagen Quick PCR cleanup , following manufacturer' s Panel C shows that in the case of cerebellar Golgi cells , instructions ( iagen , Valencia , Calif .) . there is a great deal of overlap between eGFP -L10a expres - 45 Most primer sequences ( Table 8 ) for qRT- PCR were sion in the BACarray line and expression of the genes obtained from Primer Bank (Wang and Seed , 2003 ) . PCR chosen for this analysis . This overlap confirms the specific was performed using Bio -Rad iQ syber green supermix ity of the results obtained for this and other cell types . following manufacturer ' s protocols (Biorad , Hercules, Ca) , Nonetheless , the enrichment of a particular mRNA in the IP 50 with 500 nm final concentration of each primer . Cycling and sample cannot be used to conclude that it is exclusively quantitation were performed using Biorad iQ5 multiplex expressed in the cell type labeled in the BACarray trans real- time detection hardware. PCR was carried out for 45 genic line , or that it is expressed in all cells of that type. For cycles ( 94° , 30 seconds, 63° , 30 seconds, 72°, 30 seconds) , example , the ISH databases ( available on the world wide 55 followed by a melt curve. Each replicate was assayed in web at stjudebgem .org and the world wide web at brain - triplicate . Conditions yielding dimers , as demonstrated by map .org ) clearly indicate that Penkl is expressed in scattered melt curve and /or gel electrophoresis were excluded from cells in both the granular and molecular layers of the adult further analysis . Primers that did not yield product in at least cerebellum . Furthermore , as shown in panel 1 , PenklmRNA 60 2 of 3 replicates prior to 35 cycles were excluded from does not appear to be expressed exclusively in those further analysis . Data were normalized to B -actin ( Over expressing Grm2. Finally , some mRNAs enriched in the bergh et al. , 1999 ) with the ddCT method , via iQ5 ' s optical BACarray data collected from Golgi cells were not detected system software version 2 , and averaged across replicates . using the fluorescence ISH technique , perhaps reflecting 65 All qPCR products were subcloned and sequenced to con limited sensitivity of ISH for low expressed genes or the firm accuracy of PCR . Microarray data were also normalized need for more rigorous probe design . Thus, although a clear to B - actin for comparison purposes . US 9 ,816 ,096 B2 57 58 TABLE 8 List of CRT- PCR primers CRT - PCR primer sequences were mostly from PrimerBank (Wang and Seed , 2003 ) or literature . All are listed in 5 ' to 3 ' direction . Table 8 discloses the " Forward Primer " sequences as SEQ ID NOS 16 - 29 , all respectively , in order of appearance . Gene Forward Primer Reverse Primer Primerbank ID or Ref B - Actin agagggaaatcgtgcgtgac caatagtgatgacctggccgt ( Overbergh et al . , 1999 ) Gfap gtaaagactgtggagatgcgggatggtg gtgctggtgtgggtgggaactga Chat ccattgtgaagcggtttggg gccaggcggttgtttagataca 26338049a1 Slc18a3 gtgaagataggcgtgctatttgc gactgtggaggcgaacatgac 11096330a2 Cnp1 tgcttgatgataccaaccacg gctgggcacagtctagtcg 6753476a3 Pcp2 tgcagggcgat cggatggaggag tgaggggtgagcaggggttgagg Rilp ctgatgcggcaacctcagat ttgagcaagaacacgttggct 30185833 a1 Popdc3 tgactgaacacccactctgc actgccacccataaaacctact 31745187a1 Kcnn1 ttgaaaagcgtaaacggctca cagagcaaaagagcagagtga 14161696a1 Foxqi aaattggaggtgttcgtccca tccccgtctgagcctaagg 31560693a1 Cox7 al gctctggtccggtcttttage gtactgggaggtcattgtcgg 6753504a1 Tpm2 aagtcgctgatagcctcagag ggtctggtgtatctccacgttc 50190a1 Crygs cagacttccgctcgtacctaa tcgccctggggtaagatgt 6753532 a1 Cfi cttggctctccacttgagttc ggagcgatgcgtgtatttctg 6671744a1

Example 7 clearly tightly clustered , many neuronal types ( e. g . Purkinje cells ) are not strongly clustered with any other cell type . This Comparative Analysis Of BACarray Data Collected 35 suggests that comparative analysis of BACarray transla From Many Cell Types tional profiles obtained from highly specialized cell types may yield insights into their biochemical properties . Finally , Previous examples show that the BACarray data accu individual cell types did not generally cluster tightly with the rately reflect expression of known positive and negative UB samples from their tissue of origin . In fact , profiles from controls for each cell type and that these results can be 40* UB samples derived from different brain regions were confirmed by independent experimental analysis (Heiman et loosely clustered together relative to the data obtained from al. ) . This example illustrates the broad properties of these specific CNS cell types, suggesting that microarray data cells that could be inferred from comparative analysis of this produced from dissected whole brain regions are less infor large set of microarray data . The results of this analysis are 15 mative than BACarray analysis of individual cell types. presented in FIG . 19 . A hierarchical clustering of the To examine this point in more detail, the microarray data GCRMA normalized data from all 24 IP and 6 UB samples from total cerebellum to that of the cerebellar cell types using the 20 % of probesets with the highest coefficient of analyzed in this study were compared . As can be seen in variation was performed , as presented in Panel A . This Panel B , any single cell type has fewer probesets detectable unsupervised clustering essentially recapitulates the known 50 than the whole cerebellar sample , since the whole cerebellar biology of CNS cell types . Thus , the three populations of sample represents an aggregate of different cell types . How cortical projection neurons are more similar to one another ever, comparative analysis of the sum of the probesets than they are to cortical interneurons , Purkinje cells , or detectable in each of the six individual cerebellar cell types motor neurons. Astroglial BACarray data collected from and the results obtained from whole cerebellar tissue reveals different regions of the brain are , as expected , more similar 55 over 4000 probesets that are undetectable in the microarray to one another and to Bergmann glia than they are to from whole cerebellum . These undetectable probesets tend oligodendrocytes . Oligodendroglia are more similar to each to represent cell - type enriched genes. In fact , for rare cell other than they are to any neuronal population , etc . These types , up to 42 % of the genes enriched in that cell may not findings support the concept that cells with similar gene be detectable at all in whole - tissue microarray studies . For expression patterns share similar functions , and suggest that 60 detection of genes expressed in specific cell types within analysis of BACarray data will allow for the identification of complex brain regions, therefore , the translating ribosome those gene products responsible for the distinguishing char - affinity purification ( TRAP ) methodology can be more sen acteristics of each cell type . sitive than microarray analysis of dissected brain regions. The diversity of translational profiles across neuronal The increased sensitivity of the translating ribosome types nearly rivals the diversity between neurons and glia . 65 affinity purification ( TRAP ) methodology results in identi Although related cell subtypes , such as different motor fication of more mRNAs in each cell type , yielding a more neurons or the Drdl and Drd2 medium spiny neurons, are complete picture of the translational profile for each cell US 9 ,816 ,096 B2 59 60 type , more information . To assess if this increased sensitivity 2005 ). According to this analysis , cell type diversity in the in fact does gives better information , the Shannon entropy nervous system is driven primarily by the expression of cell was calculated for each probeset across the six whole tissue surface proteins , such as channels and receptors , and also to samples, and across the twenty four individual cell popula some extent by the specific expression of transcription tions (Fuhrman et al ., 2000 ; Shannon and Weaver, 1969 ) . 5 factors and calcium binding proteins. Genes with less infor Shannon entropy is a measure of information content that mation content tend to be those that that are more ubiqui describes the complexity of a signal across samples , with tously expressed , such as ribosomal and mitochondrial pro values ranging from 0 ( low information ) to 2 (high infor - teins . This is not to say that they do not vary — their mation ) . Data are presented in FIG . 20 . Examples of probe - expression can range often from two to five fold across cell set with low and high information are shown in Panel B . 10 types — but they vary less dramatically than the tens to Shannon entropy measures of the information content in thousands fold changes of many receptors and channels . these samples reveals that the average Shannon entropy in Table 9 is a comparative analysis of translational profiling cell type specific experiments ( IP ' s ) is over twice as high as to identify co -regulated genes that could encode the highly that calculated from microarray data of whole tissue samples specialized properties of individual cell types. This analysis ( t - Test, p < 0 .0001 , average entropy across all IPs: 0 . 88 + / - 15 is can be useful in trying to identify gene sets for known 0 .002 , whole tissue : 0 .41 + / - 0 .003 ) . This analysis demon - functions or candidate functions for novel gene products , strates that microarray data collected from specific cell types since in many cases these cohorts of co -regulated genes will using the BACarray strategy can provide better information include genes with well known functions . To test whether than traditionalmicroarray studies of dissected brain tissues. the BACarray data provided can yield productive results in As those genes with high entropy measures across all 20 this sort of query , a probeset for a gene known to be involved samples are those that vary in the most complex manner in myelination was selected — the myelin basic protein between cell types , this entropy measure was applied to (Mbp ) . Its highest correlates were examined across all IP and assess what fundamentally determines the differences UB samples. In the top 35 genes correlating with Mbp between cell types in the nervous system . As seen in Panel expression (min correlation , 0 . 86 ), 6 genes also known to be C , the ten percent of the probesets with highest entropy and 25 involved in myelination were identified , including Plpi , those with the lowest entropy were classified with Gene Cnp , Mog , Mal and Mobp , and another three genes previ Ontologies and then searched for functional categories that ously identified in a proteomic screen ofmyelin components were over - represented ( Ashburner et al ., 2000 ; Maere et al ., ( Table 9 ) . TABLE 9 Genes correlating with myelin basic protein . Correlation of genes with a Mbp probeset (1419646 _ a _ at) identifies known and putative novel myelination genes. Four of the first eleven genes correlated with Mbp represent genes for the known myelin components Mal, Plp , Mobp , and Mog . This coexpression suggests that novel genes highly correlated with Mbp (C11orfy , A330104H05 Rik , Bcas1) may also be involved in myelination . Pearson ' s correlation . Only the first probeset for each gene is shown for those genes with multiple probesets on the array. In bold are genes identified in an independent proteomic screen of myelin components ( Vanrobaeys et al. , 2005 ) . Probeset Correlation Symbol Name 1419646 _ a at Mbp myelin basic protein 1417275 at 0 .968 Mal myelin and lymphocyte protein , T - cell differentiation protein 1451718 at 0 . 964 Plp proteolipid protein (myelin ) 1440902 at 0 . 958 Galnt5 UDP - N - acetyl- alpha - D galactosamine: polypeptide N - acetylgalactosaminyltransferase 5 1433785 at 0 . 958 Mobp myelin -associated oligodendrocytic basic protein 1436578 at 0 . 955 A330104H05 Rik novel 1428792 _ at 0 . 946 Bcas1 breast carcinoma amplified sequence 1 1439506 _ at 0 . 934 C11ORF9 novel homolog 1426960 _ a __ at 0 . 931 Fa2h fatty acid 2 -hydroxylase 1433543 _ at 0 . 926 Anln anillin , actin binding protein ( scraps homolog , Drosophila ) 1448768 _ at 0 . 921 Mog myelin oligodendrocyte glycoprotein 1418086 _ at 0 . 917 Ppplr14a protein phosphatase 1 , regulatory ( inhibitor) subunit 14A 1434094 _ at 0 . 914 6330530AO5Rik novel 1420760 _ S _ at 0 . 911 Ndrg1 N -myc downstream regulated 1 1447807 _ s _ at 0 . 905 Plekhh1 pleckstrin homology domain containing , family H (with MyTH4 domain ) member 1 1425546 _ a _ at 0 . 905 Trf transferrin 1436974 _ at 0 . 904 A230069A22 Rik novel 1450241 _ a _ at 0 . 904 Evi2a ecotropic viral integration site 2a 1418472 _ at 0 . 903 Aspa aspartoacylase (aminoacylase ) 2 1452834 _ at 0 . 9 2600010E01 Rik novel 1429909 _ at 0 . 894 4833411004Rik novel 1453009 _ at 0 . 893 1110060101Rik novel 1440813 _ s _ at 0 . 89 Plxnb3 plexin B3 1437171 _ x _ at 0 . 888 Gsn gelsolin 1423871 at 0 . 887 BC014795 novel US 9 ,816 , 096 B2 61 62 TABLE 9 - continued Genes correlating with myelin basic protein . Correlation of genes with a Mbp probeset ( 1419646 _ a _ at ) identifies known and putative novel myelination genes. Four of the first eleven genes correlated with Mbp represent genes for the known myelin components Mal, Plp , Mobp , and Mog . This coexpression suggests that novel genes highly correlated with Mbp (C11orf9 , A330104H05Rik , Bcas1 ) may also be involved in myelination . Pearson ' s correlation . Only the first probeset for each gene is shown for those genes with multiple probesets on the array. In bold are genes identified in an independent proteomic screen of myelin components (Vanrobaeys et al ., 2005 ) . Probeset Correlation Symbol Name 1434399 at 0 .887 Galnto UDP - N -acetyl - alpha - D galactosamine :polypeptide N -acetylgalactosaminyltransferase 6 1435854 _ at 0 . 885 Tmem10 transmembrane protein 10 1416371 at 0 . 878 Apod apolipoprotein D 1418406 _ at 0 . 877 Pde8a CAMP- specific cyclic nucleotide phosphodiesterase PDE8 1416003 _ at 0 .875 Cldn11 claudin 11 1434606 _ at 0 .872 Erbb3 V - erb -b2 erythroblastic leukemia viral oncogene homolog 3 ( avian ) 1451932 _ a _ at 0 . 872 Tsrc1 thrombospondin repeat containing 1 1418980 _ a _ at 0 . 871 Cnp1 cyclic nucleotide phosphodiesterase 1 1416318 _ at 0 .87 Serpinbla serine (or cysteine ) proteinase inhibitor, clade B , member la 1424468 _ s _ at 0 . 868 D330037A14Rik novel

25 FIG . 21 shows a comparative analysis of the individual ating in these cell types , or for the identification of new cell samples to identify only those genes most highly proteins operating in well known pathways . Finally, com specific for each population . An iterative comparison was parative analysis can reveal discrepancies that are not appar performed : one -by -one , each sample was compared to each ent from anatomical studies . For example , the most specific other sample in the dataset, and for each population , probe - 30 probesets for the Etvl line identify several genes well sets were sorted by their average ranking across these known to be expressed in lymphoid cells , suggesting that in comparisons. Data were then combined and clustered by this line the eGFP -L10a transgene may also be expressed in expression the top one hundred ranked probesets for each circulating cells in the CNS vasculature . Taken together, the population in a heatmap (Panel A ). This heat map readily data shown above demonstrate two important strengths of illustrates the extent to which distinct cell types are charac - 35 large - scale comparative analyses of BACarray data . First , terized by specific cohorts of genes . For example , cerebellar molecular relationships between cell types can be easily Purkinje cells are clearly distinguished by a group of genes established with hierarchical clustering ; second , groups of that are not seen in any other cell types ( Panel A ) . Thus none genes that encode the biochemical functions of specific cell of the top twenty five most specific probesets observed in the types can be identified using this sort of systematic com Purkinje cell sample are found in any of the top twenty five 40 parative approach . most specific probesets for any of the other cell types . In contrast , Drd1 and Drd2 medium spiny neurons, two closely Example 8 related cell types , co - express many genes that are not found in the other cell populations analyzed , yet they also express Analysis of BACarray Data Collected from Spinal distinct subsets of genes that differentiate them (Heiman et 45 Motor Neurons al) . Thus , comparative analysis of BACarray data can be used to characterize CNS cell populations with unique Due to their involvement in a variety of serious neuro biochemical and physiological properties, and to distinguish logical disorders and severe , acute injuries , spinal cord between closely related cell types at the molecular and motor neurons (MN ) are among the most well studied cell biochemical level. 50 types of the CNS . As such , they provide an opportunity to As shown in the tables in Panel B , the top twenty five evaluate the BACarray data with the depth of the knowledge most specific probesets in each cell type include probesets available for this cell type . In particular, a wealth of ana for both well known cell -specific markers and novel, pre - tomical and physiological data available for MN , and the viously uncharacterized genes. For example , Pcp2 , the cal comprehensive studies of transcription factors involved in cium binding protein Calb1, the scaffolding / synaptic protein 55 their development, allowing comparison of the BACarray Homer3 , and the transcription factor Ebf2 , all of which are data presented here with the published literature . As shown known to be specifically expressed in Purkinje cells (Mal in FIG . 22 , in a single BACarray experiment, most of the garetti et al ., 1997 ; Shiraishi et al. , 2004 ; Wang et al. , 1997 ) , MN expressed molecules that have been documented in are among the most highly ranked probesets in the Pcp2 prior studies are rediscovered . To perform this analysis , BACarray list . Mobp , one of the most abundant components 60 BACarray results were color coded as expressed , of the CNS myelin sheath (Montague et al. , 2006 ) , is “enriched , ' or ‘not expressed , ' as described in the methods . prominent in the Cmtm5 myelinating oligodendrocytes ' list. This classification was then compared to results reported in The expression of Torb in deep layer cortical neurons the adult rodent literature , color coded simply as either (Nishiyori et al. , 2004 ) is confirmed in the Ntsr1 BACarray expressed ' or ' not expressed or left uncolored in cases data . The large number of uncharacterized genes with cell 65 where there were no studies or conflicting data. In most specific translation identified here provide an important cases , where microarray probesets were present and infor resource for discovery of novel biochemical pathways oper - mative , BACarray results agree well with the literature . US 9 ,816 ,096 B2 63 64 Thus , it has been reported that MNs express glutamate characteristic Purkinje cell morphology ( Panel c ) . Restric receptors sensitive to AMPA , kainate , and NMDA (Rekling tion of eGFP -L10a expression to cerebellar Purkinje cells et al. , 2000 ) . These results suggest that the specific receptor was confirmed by indirect immunofluorescent staining of subunits mediating these responses include Gria3 and 4 , Calbindin - D28K ( Panel D ) , which in the cerebellum is Grik2 and 4 , and Grin1 , 3a and 3b . Inhibition in MNs should 5 specifically expressed in Purkinje cells (Nordquist et al. , be due the actions of the Glra2 and GirB glycine receptor 1988 ) . Array data were collected from brain stem cholin subunits and both metabotropic (Gabbrl ) and ionotropic ergic motor neurons using the Chat BACarray line and from GABAergic receptors , potentially composed ofGabra2 , a5 , Purkinje cells using the Pcp2 BACarray line. Replicate Chat and 33 subunits . These data predict that MNs should BACarray samples gave nearly identical genome- wide respond to all classic neurotransmitters , including acetyl- 10 choline , via Chrnc4 /62 and / or Chrna7 receptors , and sero translational profiles (average Pearson correlation = 0 . 982 ), tonin , via the Htrld receptor. In disagreement with prior as was the case for replicate Pcp2 immunohistochemical findings (Rekling et al ., 2000 ), BACarray samples ( average Pearson correlation = 0 .997 ) . expression of Drdl and or Drd2 in MNs was not detected . To provide a measure of the enrichment of each mRNA Moreover , transgenic mice for Drdl and Drd2 do not show 15 immunoprecipitated from the targeted cell type ( IP ) versus transgene expression in MNs, nor does the Allen Brain Atlas its expression in a reference sample ( unbounds in immuno ISH show expression in brain stem MN , supporting the precipitations ), the ratio of the expression in each IP sample BACarray results . versus the reference sample was calculated . This compari MNs also express a variety of newly characterized recep son identified those genes which were highly enriched in tors and orphan receptors . For example , BACarray data has 20 each cell type versus a common reference sample . As successfully identified Grin3b as a MN specific gene encod expected from the analysis of D1 and D2 combined BAC ing an NMDA subunit . This receptor was recently charac array data versus whole brain minus, differences are evident terized as creating a unique glycine gated channel in MNS between the genome- wide translational profiles of IP ( Chatterton et al . , 2002 ; Nishi et al. , 2001) . Several other samples compared to the reference sample for all the indi genes enriched in MNs have also been identified which 25 vidual samples analyzed : striatonigral (D1 ) , striatopallidal potentially encode for MN specific receptors that either have (D2 ), brain stem cholinergic ( Chat) , and Purkinje cells not been previously characterized in MNs or are entirely (Pcp2 ) . FIG . 24 shows that enrichment of cell - specific unstudied . Two that are particularly interesting are the positive - control genes and exclusion of known negative vitamin D receptor and the orphan receptor P2rx11 . Future control genes ( glial genes ) , were evident for each compari studies investigating the role of these receptors in MN 30 son ( panels a - d ) . Venn diagrams constructed from the top behavior may explain cases of reversible muscle weakness 1 ,000 enriched probesets from this analysis ( Tables 17 - 20 ) in patients with vitamin D deficiency (Whitaker et al. , 2000 ; confirmed that the translational profiles of striatonigral and Ziambaras and Dagogo - Jack , 1997) , or suggest new path striatopallidal cells show greater similar to each other than ways important to MN function . they do to the translational profiles of either brain stem 35 cholinergic cells or Purkinje cells (panels e- h ). Example 9 Example 10 Molecular Phenotyping and Translational Profiling of Cholinergic Motor Neurons and Purkinje Molecular Phenotyping and Translational Profiling Neurons 40 of the Striatum To determine whether the BACarray technique could be 1 ) Overview used for the characterization of other types of neurons , This example presents a study of the specific properties of cholinergic and Purkinje cell - specific BACarray lines were the striatum . This example illustrates one embodiment that produced , as presented in FIG . 23 . The cholinergic cell 45 can uncover unexpected molecular and physiological com BACarray line was produced by placement of the eGFP - L plexity in closely related and spatially adjacent CNS cell 010a transgene under the control of the choline acetyltrans - populations . The striatum is a subcortical part of the tel ferase ( Chat) locus, which is specifically expressed in cho encephalon . It is the major input station of the basal ganglia linergic cells in the CNS . As expected from the published system . In this example , transgenic mice containing molecu Chat expression pattern in the rat CNS (Oh et al. , 1992 ) , the 50 larly tagged ribosomal proteins in the striatonigral and Chat BACarray line DW167 showed highest eGFP -L10a striatopallidal cells of the striatum were created using stan expression in cholinergic cells of the dorsal striatum and dard BACarray techniques . Striatonigral and striatopallidal ventral striatum (nucleus accumbens , olfactory tubercle , and medium spiny neurons (MSNs ) , are intermixed , indistin islands of Calleja ), basal forebrain , brain stem , spinal cord , guishable in somato - dendritic morphology, and of major and medial habenula (Panel a ) . eGFP expression was shown 55 interest due to their role in the etiology of various neuro to be restricted to cholinergic cells in all of these structures , logical diseases, including Parkinson ' s disease , schizophre including motor neurons of the brain stem ( Panel b ) , by nia , attention deficit hyperactivity disorder, drug addiction , indirect immunofluorescence staining for Chat. One excep - and Huntington ' s disease . Striatonigral MSNs send projec tion to this colocalization was in the pedunculopontine and tion axons directly to the output nuclei of the basal ganglia , laterodorsal tegmental nuclei, where only a minority of 60 i . e . the substantia nigra and the internal segment of the cholinergic cells were labeled with eGFP . globus pallidus ( the entopeduncular nucleus in rodents ) , The Purkinje cell BACarray line DR166 was produced by while striatopallidal MSNS send projection axons to the placement of the eGFP -L10a transgene under the control of external segment of the globus pallidus. Striatonigral MSNs the Purkinje cell protein 2 (Pcp2 ) locus, which is specifically are known to preferentially express the dopamine D1 recep expressed in cerebellar Purkinje cells of the CNS (Oberdick 65 tor (Drd1 ; Drdla ) and the neuropeptides substance P ( Tac1) et al. , 1988 ) . The Pcp2 BACarray line showed eGFP -L10a and dynorphin (Pdyn ) , while striatopallidal MSNs preferen expression that was restricted to cells that possessed a tially express the dopamine D2 receptor ( Drd2 ) , the adenos US 9 ,816 ,096 B2 65 66 ine A2a receptor (Adora2a ) and the neuropeptide enkephalin 3 ) BACarray Profiling of Striatonigral and Striatopallidan ( Penk ) ( S . Gong et al. , Nature 425 , 917 -25 (2003 )) . MSNs 2 ) Production of BACarray Striatal Mice A plurality of differentially expressed genes can be iden Mice containing molecularly tagged ribosomal proteins in tified which characterize striatonigral and striatopallidal the striatonigral and striatopallidal cells of the striatum were 5 MSNs , including all previously known markers . Translation created . Homologous recombination in bacteria was used to profiling and molecular Phenotyping was performed with place eGFP -L10a under the control of either the D2 receptor immunoaffinity - purified mRNA from adult striatonigral or ( striatopallidal) or D1 receptor (striatonigral ) loci in the appropriate BACs. Mouse lines were generated by pronu striatopallidal BACarray mice . Following two rounds of in clear injection of engineered BACarray DNA constructs into 10 vitro transcription , biotin - labeled antisense RNA ( CRNA ) fertilized oocytes. was used to interrogate Affymetrix GeneChip Mouse Mouse lines were screened by immunohistochemistry for Genome 430 2 . 0 arrays . For each cell type , data were appropriate expression of the transgene, as judged by known collected from three independent biological replicates , each D1 and D2 receptor expression patterns. In the Drd2 line , prepared from a cohort of 7 animals . Analysis of immuno 75 . 8 % colocalization of pro - enkephalin with eGFP -L10a 15 affinity - purified samples revealed no bias for mRNA length ( n = 204 / 269 ) was observed and in the Drdla line 0 % colo or abundance (FIG . 27 ). Lengths of transcripts were based calization was observed ( n = 0 / 325 ) . For the Drd2 line , this on all available mouse curated RefSeq RNA sequences number is likely an underestimate , as heat- induced epitope (available using the file transfer protocol ncbi. nih . gov / ge retrieval in citrate buffer is needed for optimal staining using nomes/ M _ musculus /RNA ) . Where multiple transcript vari pro - enkephalin antibodies. Heat- induced epitope retrieval in 20 ants for a single gene were available , the longest one was the colocalization experiments was not utilized , however, chosen . RefSeq lengths were plotted against D1 (a ) or D2 ( b ) because both eGFP fluorescence and the ability of various BACarray IP normalized expression values . There was no GFP antibodies to recognize eGFP are substantially dimin - correlation observed between transcript length and IP val ished upon use of these epitope retrieval methods. Colocal- ues . Striatal expression values for all Affymetrix Genechip ization data for eGFP -L10a ( direct eGFP fluorescence ) and 25 probe sets were obtained by total RNA arrays from wild pro - enkephalin expression ( immunohistochemical staining type striatal tissue ( data not shown ) . These values were is presented (FIG . 25 ) : ( a ) Immunohistochemistry to eGFP plotted against ( c ) D1 BACarray or ( d ) D2 BACarray IP in adult sagittal sections from the D2 BACarray line CP101 . normalized values . As expected , higher expression in total ( b ) Characterization of D2 BACarray line CP101 striatal striatum ( no IP , wild type mice ) correlates with higher D1 or MSN cells: direct eGFP fluorescence ( left panel with high - 30 D2 BACarray IP values . The few genes that showed modest magnification image insert ) ; enkephalin immunohisto - expression in total striatum but had low IP values include chemical staining (middle panel) ;merge ( right panel, with known non -neuronal genes. 20 m scale bar ) . ( c ) Immunohistochemistry to eGFP in adult Comparative analysis of these data revealed that all 8 of sagittal sections from the D1 BACarray line CP73 . ( d ) the well- characterized , differentially expressed MSN mark Characterization of D1 BACarray line CP73 striatal MSN 35 ers were enriched using the BACarray approach : D2 (Drd2 ) cells : direct eGFP fluorescence (left panel ) ; enkephalin ( 36 .6x ) , adenosine 2a receptor ( Adora2a ) ( 13 .2x ) , and immunohistochemical staining (middle panel) ; merge ( right enkephalin (Penk ) ( 7 .5x ) were enriched in the striatopallidal panel) . BACarray sample , while D1 (Drdla ) (3 .9x ), substance P The D2 BACarray line showed highest transgenic eGFP ( Tac1) (3 .6x ), and dynorphin (Pdyn ) ( 5 .6x ) were enriched in L10a expression in the dorsal and ventral striatum , olfactory 40 the striatonigral BACarray sample ( FIG . 28 and tubercle , and hippocampus. In addition , expression of eGFP Table 10 ) . Four striatopallidal - enriched mRNAs (Adk , was seen in the substantia nigra pars compacta and ventral Plxdc1, BC004044 , and Hist1h2bc ) and six striatonigral tegmental area of this line, as expected due to D2 autore - enriched mRNAs ( Slc35d3 , Zfp521, Ebf1 , Stmn2, Gnb4 , ceptor expression in dopaminergic cells (panel a ) . The D1 and Nrxnl ) were confirmed as reported in a microarray BACarray line showed highest transgenic eGFP -L10a 45 study of FACS sorted MSNS ( S . Magdaleno et al ., PLoS Biol expression in the dorsal and ventral striatum , olfactory bulb , 4 , e86 (2006 ) ) . However , the data identified approximately olfactory tubercle , and cortical layers 5 and 6 (panel c ). As 70 additional striatopallidal enriched transcripts and over expected for a ribosomal protein fusion , direct visualization 150 additional striatonigral- enriched transcripts of eGFP fluorescence revealed localization of transgenic Table 10 ) . To provide an initial test of the data , quanti eGFP -L10a to the nucleoli and cytoplasm ( panel b ) . eGFP 50 tative PCR studies were carried out using independent co - localization with enkephalin expression (striatopallidal biological BACarray D1 and D2 samples and a different cell marker ) was observed in striatal cells from the D2 cDNA amplification procedure ( A . Alexa , J . Rahnenfuhrer, BACarray line but not the D1 BACarray line (panels b and T . Lengauer , Bioinformatics 22 , 1600 -7 (2006 )) . Differential d ), verifying correct BAC -mediated cell - type expression . expression of Eyal, Isli , Gng2 , and Crym in striatonigral The polysome profile from D2 BACarray mouse striatal 55 MSNs, and Gpro , Lhx8 , Gpr88 , Trpc4 , and Tpm2 in stri extract is presented in FIG . 26 as follows : Top : Post - atopallidalMSNs was confirmed ( Table 11 and Table 12 ) . mitochondrial striatal extract (S20 ) was loaded onto a linear qPCR assays used were Gapdh : Mm99999915 gi , Drd2 : sucrose gradient ( 20 - 50 % w / w ). After velocity sedimenta Mm00438541 _m1 , Gpr6 : Mm01701705 _ sl , Lhx8 : tion , fractions (direction of sedimentation noted by arrow ) Mm00802919 _m1 , Gpr88 : Mm02620353 _s1 , Trpc4 : were collected as UV absorbance ( 254 nm ) was measured . 60 Mm00444284 _m1 , Tpm2: Mm00437172 _ g1 , Eyal: Bottom : gradient fractions were ethanol precipitated , resus - Mm00438796 _ m1, Tacl : Mm00436880 _ml , Isl1 : pended in SDS - PAGE loading buffer , and Rp17 and eGFP - Mm00627860 _ml , Gng2 : Mm00726459 si , Chrm4 : L10a content were assayed by Western blotting . Velocity Mm00432514 _ sl, Drdla : Mm02620146 _ s1, Crym : sedimentation analysis of polysome complexes isolated Mm01281258 _ m1, Actb : Hs99999903 _ml , and Fth1 : from striatal extracts for both lines of BACarray mice 65 Hs01694011 _ s1 . Each assay was performed in quadrupli confirmed incorporation of the eGFP - L10a fusion protein cate and fold enrichment values were derived from the into functional polysomes in vivo . comparative Ctmethod ( following Applied Biosystems rec US 9 ,816 ,096 B2 67 68 ommendations) , with each target amplification compared to TABLE 10 -continued a Gapdh or Actb reference amplification . Genes differentially translated between TABLE 10 striatonigral and striatopallidal cells . Probe Symbol Genes differentially translated between GenBank FCH striatonigral and striatopallidal cells . 1427510 _ at Gnail U38501 3 . 16 1436862 at Doc2a BB543070 3 . 13 Probe Symbol GenBank FCH 1452065 _ at Vstm2 BB085570 3 .11 Rassf3 3 .08 Eyal 1448546 _ at BB703307 1457424 _ at BB760085 32 . 49 10 1429175 _ at Tmem178 AK014196 3 . 08 1424606 _ at Cplx3 BC024854 19 . 56 1418417 _ at Msc NM _ 010827 3 . 08 1418047 _ at Neurodo NM _ 009717 13 .05 1448812 at Hpcali NM 016677 3 .07 1428393 _ at Nrn1 AK003046 12 . 21 1454691 at Nrxnl BB336165 3 .04 1417560 at Sfxn1 BB478992 11 . 99 1450042 _ at Arx BB322201 3 . 04 1422720 _ at Isli BQ176915 10 . 10 1446498 _ at I120ra BB551879 3 . 03 1450723 _ at Isli BO176915 9 . 44 15 1457984 _ at Crh BM933756 3 .03 1420388 at Prss12 NM 008939 9 . 25 1438729 _ at BB176347 BB331017 3 . 03 1419473 _ a at Cck NM 031161 9 . 11 1440108 _ at Foxp2 BM964154 2 .99 1447863 _ s _ at Nr4a2 BB322941 9 . 00 1434695 _ at Dtl AV270035 2 .97 1419200 _ at Fxyd7 NM _ 022007 7 . 90 1439632 _ at BQ174397 2 . 96 1455034 _ at Nr4a2 BB703394 7 . 52 1417288 _ at Plekha2 NM 031257 2 . 93 1417559 _ at Sfxn1 BB478992 7 .42 1426937 _ at 6330406115 Rik AK018128 2 . 92 1428642 _ at Slc35d3 AK018094 7 . 32 20 1438624 _ x _ at Hs3 st2 BB494451 2 . 85 1451499 _ at Cadps2 AF000969 6 . 87 1440849 _ at 6330417G04Rik AV327187 2 .85 1450750 _ a _ at Nr4a2 NM 013613 6 . 44 1426738 _ at Dgkz BC014860 2 .84 1426852 _ xat Nov X96585 6 . 44 1428781 at Dmkn BI452905 2. 83 1431339 _ a _ at Efhd2 AK007560 6 .00 1439789 _ at Ebfi BQ177189 2 . 83 1428184 _ at 3110035E14Rik BB348639 5 .61 1450055 _ at Vsnl1 NM 012038 2 . 83 1416266 _ at Pdyn AF026537 5 . 59 25 1417569 _ at Ncald BG071381 2 .80 1428157 _ at Gng2 AVO21455 5 . 08 1437671 _ _ x _ at Prss23 BB378796 2. 79 1441388 _ at BB428710 5 .05 1444661 at Gpr26 BB247791 2 .79 1437478 _ s _ at Efhd2 AA409309 5 . 04 1427975 at Rasl10a AK008807 2 . 77 1423280 _ at Stmn2 BM115022 4 . 89 1438112 _ at 9430021MO5Rik AA546727 2 . 76 1428156 _ at Gng2 AVO21455 4 . 85 1436787 _ x _ at Sec1413 AVO24133 2 .73 1421727 _ at Eyal NM __ 010164 4 . 84 30 1417579 _ x _ at Gmppa NM 133708 2 .72 1451322 _ at Cmbl BC024580 4 . 82 1456146 _ at 2210411 A11 Rik BI735554 2 . 72 1423281 _ at Stmn2 BM115022 4 . 73 1436503 at BC048546 BF302511 2. 71 1448293 at Ebfi BB125261 4 . 72 1420720 _ at Nptx2 NM _ 016789 2 .70 1416711 _ at Tbr1 NM _ 009322 4 . 70 1455925 _ at Prdm8 AV349236 2 . 68 1422256 _ at Sstr2 NM _ 009217 4 .68 1448632 _ at Psmb10 NM _ 013640 2 .67 1416302 _ at Ebfi BB125261 4 . 55 35 1426329 _ s _ at Baalc AF371320 2 .65 1440361_ at BB272510 4 . 48 1448669 _ at Dkk3 AK004853 2 .65 1455893 _ at Rspo2 BG067392 4 . 35 1425090 _ S _ at Konc4 BC024837 2 .64 1428664 _ at Vip AKO18599 4 . 35 1454959 _ s _ at Gnai 1 BO174580 2 .63 1419470 at Gnb4 BI713933 4 .32 1453558 _ at 4930504H06Rik AK015697 2 .63 1452779 _ at 3110006E14Rik AKO14009 4 . 26 1423757 _ x _ at Igfbp4 BC019836 2 .62 1416301 _ a _ at Ebfi BB125261 4 .25 1437405 a at Igfbp4 BB787243 2 .59 1436312 at Ikzfi AV317621 4 . 20 40 1450059 _ at Fancg BG072083 2 .54 1424737 _ at Thrsp BC009165 4 . 16 1450708 _ at Scg2 NM _ 009129 2 . 52 1448366 _ at Stxla NM _ 016801 4 . 14 1429269 at BC068157 BE992549 2 .51 1451620 _ at C1q13 BB768838 4 . 11 1422537 _ a _ at Id2 NM _ 010496 2 . 51 1448889 _ at Slc38a4 NM 027052 4 .08 1417568 _ at Ncald BG071381 2 .49 1426851 _ a _ at Nov X96585 4 .07 1435411 at BE950834 2 . 49 1426318 at Serpinb1b AF426025 4 . 03 45 1424966 _ at Tmem40 BC019416 2 .48 1423287 _ at Cbln1 AA016422 4 .01 1438796 _ at Nr4a3 BE692107 2. 48 1417343 at Fxyd6 AB032010 3 . 99 1416776 _ at Crym NM 016669 2 . 47 1427017 _ at Satb2 BB104560 3 . 92 1437406 _ x _ at Igfbp4 BB787243 2 . 45 1419469 _ at Gnb4 BI713933 3 . 89 1423202 _ a _ at Ncor1 U22016 2 . 45 1455629 _ at Drdla BE957273 3 . 87 1444307 _ at AW491448 2 . 44 1417400 _ at Rai14 NM 030690 3 . 74 50 1439573 _ at Rtn4r12 BE992565 2 .43 1423286 _ at Cbln1 AA016422 3 . 71 1417605 _ s _ at Camk1 NM _ 133926 2 .42 1456051 _ at Drdla BB282271 3 . 69 1425784 _ a _ at Olfml D78264 2 . 39 1419184 _ a _ at Fh12 NM _ 010212 3 . 64 1455074 _ at Efcab1 AV307860 2. 39 1434440 _ at Gnail BQ174580 3 .64 1451342 _ at Spon1 BC020531 2 . 38 1418789 _ at Sntg2 NM _ 133742 3 .61 1420955 _ at Vsnl1 NM _ 012038 2 . 38 Tac1 NM _ 009311 3 .61 1416783 _ at 55 1417192 _ at Tomm70a NM _ 138599 2. 38 1457440 _ at Sstr4 BB451927 3 .59 55 1452169 _ a _ at Dgkz BC014860 1418451_ at Gng2 BB522409 3 .57 1418304 _ at Pcdh21 NM 130878 2 .37 1442724 _ at AW060763 AW120464 3 .55 1454770 _ at Cckbr AV221910 2 . 37 1455758 _ at Prkcc BM215011 3 .52 1440918 at AW495511 2. 37 1430306 _ a _ at Atpovic2 AK004157 3 . 48 1448754 _ at Rbpl NM _ 011254 2 . 37 1456515 Sat Tcf15 AV044715 3 . 43 1428240 _ at Nrxnl AK017578 2 . 36 1416953 _ at Ctgf NM _ 010217 3 . 40 60 1453245 at 9130024F11 Rik BB329046 2 . 36 1441917 _ s _ at Tmem40 BB468188 3 . 35 1438231 _ at Foxp2 AV322952 2 . 36 1437091 _ at Accn4 AV323885 3 . 30 1438232 at Foxp2 AV322952 2 . 35 1436786 _ at Sec1413 AVO24133 3 .29 1417578 _ a _ at Gmppa NM 133708 2 . 34 1429274 _ at 2310010M24Rik AK009282 3 .23 1428077 _ at Tmem163 AK011522 2 .32 1421017 _ at Nrg3 NM 008734 3 . 23 1453317 _ a _ at Khdrbs3 AK014353 2 . 32 1416503 _ at Lxn NM 016753 3 .22 65 1419551 _ s _ at Stk39 BG919998 2 . 32 1417933 _ at Igfbp6 NM 008344 3 .18 1421144 _ at Rpgrip1 NM _ 023879 2 . 32 US 9, 816 ,096 B2 69 70 TABLE 10 - continued TABLE 10 - continued Genes differentially translated between Genes differentially translated between striatonigral and striatopallidal cells. striatonigral and striatopallidal cells . Probe Symbol GenBank FCH Probe Symbol GenBank FCH 1421926 _ at Mapk11 AV329330 2 . 30 1416008 _ at Satb1 AV172776 1 . 81 1415849 _ s _ at Stmn1 BC010581 2 . 30 1449839 _ at Casp3 BG070529 1 . 80 1424763 _ at 1700027N10Rik BC019423 2 . 27 1420858 _ at Pkia AK010212 1 . 79 1459847 _ x _ at Gfra2 AV354240 2 . 27 1425006 _ a _ at Vrkl BC016676 0 . 57 1449314 _ at Zfpm2 NM _ 011766 2 . 26 10 1437573 at BF018351 0 . 57 1430634 _ a _ at Pfkp BB076574 2 . 26 1456434 _ x _ at BB764222 0 . 56 1457032 _ at Ak5 BB546359 2 . 25 1452879 _ at Synpo2 A1848603 0 . 55 1453129 _ a _ at Rgs12 AK004813 2 .24 1436238 at Lgi3 A1841179 0 .54 1447669 _ s _ at Gng4 AV347903 2 . 24 1431320 _ a _ at Myo5a AK002362 0 . 54 1433909 _ at AW048713 2 .22 1448845 _ at Rpp25 NM 133982 0 .53 1424701 at Pcdh20 BB528056 2 . 21 15 1428574 a at Chn2 AK006398 0 .53 1435959 _ at Arhgap15 BM246535 2 .21 1416967 _ at Sox2 U31967 0 . 53 1417988 at Resp18 NM _ 009049 2 . 21 1445461_ at BB096245 0 . 53 1455885 _ at Gna12 AV238106 2 . 21 1426572 _ at Me2 BM235734 0 . 53 1427054 _ s _ at Abi3bp BC026627 2 . 20 1423171 at Gpr88 BE947345 0 .53 1458112 at Adarb2 BB527550 2 . 20 1423424 _ at Zic3 BB732077 0 .52 1427115 _ at Myh3 M74753 2 . 19 1451236 _ at Rerg BC026463 0 .52 1449556 _ at H2- T23 NM _ 010398 2 . 18 20 1420533 _ at dallasigriminisGucyla3 AK004815 0 .52 1435176 _ a _ at Id2 BF019883 2 . 16 1419754 _ at Myo5a NM 010864 0 . 52 1422552 _ at Rprm NM _ 023396 2 . 15 1455265 _ a at Rgs16 BB100249 0 .52 1416131 _ s _ at C920006C10Rik BB188557 1419717 _ at Semaze NM _ 011348 0 . 52 1436609 _ a _ at Lrpapl AV309553 nirinin 1436946 _ s _ at Gng5 AV216608 0 .51 1451332 _ at Zfp521 BC021376 2 .13 1419738 _ a _ at Tpm2 AK003186 0 .51 1452444 _ at Napb AU067119 2 . 13 25 1439526 _ at Me2 AV375160 0 . 51 1457277 _ at BC038925 AI314927 2 .13 1458951 _ at Vrk1 AV341598 0 .51 1434243 _ S _ at Tomm70a AV233564 2 . 12 1441368 at BB102769 0 . 50 1436592 _ at BB453760 2 . 12 1455753 at C630035NO8Rik BQ176181 0 .50 1448249 _ at Godl BC019391 2 . 11 1436994 _ a _ at Histlhlc BB533903 0 . 49 1417604 _ at Camk1 NM _ 133926 2 . 09 1428283 _ at Cyp2s1 AK004699? 0 . 49 1443129 _ at BB363699 2 . 09 30 1420694 _ a at Dach1 BB522228 0 . 49 1452741 _ s _ at God2 BQ175968 2 .08 1454821 _ at B3gat1 BB424673 0 . 49 1417960 _ at Cpeb1 NM _ 007755 2 .08 1452540 _ a _ at Hist1h2bp M25487 0 .48 1418726 _ a _ at Tnnt2 NM _ 011619 NNN2 . 08 1456935 _ at A1606473 A1606473 0 . 48 1442887 _ at Gpr39 BQ175060 2 .07 1419879 _ s _ at Trim25 AA960166 0 . 48 1421176 _ at Rasgrp1 BB354696 2 .06 1426215 at Ddc AF071068 0 . 48 1428650 _ at Tns1 AK003780 2 . 06 35 1420534 _ at Gucyla3 AK004815 0 . 48 1428323 _ at Gpd2 BQ175968 2 .06 1418072 _ at Hist1h2bc NM _ 023422 0 .47 1425287 at Zfp189 BC021326 2 . 06 1426573 at Me2 BM235734 0 .47 1452974 _ at Nol8 AK017551 2 . 05 1437231 at Slitrko AV246497 0 . 47 1417038 _ at 8 - Sep NM _ 017380 2 .03 1456858 _ at Gpr149 BB075339 0 .47 1417943 at Gng4 NM _ 010317 2 .03 1442226 _ at Sema3e AV348197 0 . 47 1448326 _ a _ at Crabp1 NM _ 013496 2 .03 1448839 at Ankrd47 NM _ 030697 0 . 46 1416407 _ at Pea15 A1323543 2 .03 40 1422673 at Prkcm NM _ 008858 0 .46 1436821 _ at Plcxd3 BB293136 2 .02 1443365 _ at Htr4 BB431008 0 . 46 1426606 _ at Crtac1 BB426194 2 .01 1438841 _ s _ at Arg2 AV002218 0 . 46 1449429 _ at Fkbp1b NM _ 016863 2 .01 1434141 _ at Gucyla3 BG072799 0 . 46 1452731 _ x _ at LOC554327 BM195235 2 .00 1448415 _ aat Sema3b NM _ 009153 0 .45 1425854 _ x _ at Terb - V13 U07661 2 .00 1439614 at Htr4 BB308379 0 . 45 1452936 _ at Crtac1 AV341285 1 . 99 45 1436913 at Cdc14a BB479310 0 . 45 1448660 _ at Arhgdig NM _ 008113 1 .98 1451033a at Tpc4 BB271442 0 . 45 1415897 _ a _ at Mgst1 BI150149 1 . 98 1449241 at Klhli NM 053105 0 . 45 1431429 _ a _ at Arl4a AK006286 1 . 98 1460327 _ at Gpr88 BE947345 0 .45 1416004 _ at Ywhah NM __ 011738 1 . 97 1448162 _ at Vcam1 BB250384 0 . 44 1418982 _ at Cebpa BC011118 1 . 97 1446681 _ _ at BB086117 AV327329 0 . 44 1418271 _ at ?hlhb5 NM _ 021560 1 . 96 50 1450992 _ a _ at Meis1 AW547821 0 . 44 1449403 _ at Pdega NM _ 008804 1 . 95 1449577 _ x _ at Tpm2 AK003186 0 . 44 1449799 _ s _ at Pkp2 AA516617 1 . 94 1417688 _ at BC004044 NM 030565 0 . 44 1454745 _ at Arhgap29 BG074320 1 . 94 1435138 _ at Tmem28 AV016797 0 .43 1425051 _ at Isocl AK010892 1 .94 1457648 _ x _ at BC004044 BB084182 0 .42 1455324 _ at Plcxd2 BQ176176 1 . 94 1417306 _ at Tyk2 NM _ 018793 0 . 42 1418792 _ at Sh3g12 AF326561 1 . 93 55 1424854 _ at Hist1h4i BC019757 0 . 41 1437390 _ x _ at Stxla AV339210 1 .93 1443827 __ x _ at BC004044 BB375974 0 .41 1416133 at C920006C10Rik BB188557 1 .93 1418895 _ at Skap2 NM 018773 0 . 41 1455443 _ at Gdapili BB733286 1 .93 1443372 _ at BB417900 0 . 41 1433664 at Ube2q2 BM202586 1 . 92 1440484 at Unc5d BB525782 0 . 41 1431749 _ a _ at Rasgrp1 AK013548 1 . 91 1448276 _ at Tspan4 NM _ 053082 0 . 40 1455374 _ at BB332873 1 . 91 1425028 _ a _ at Tpm2 BC024358 0 . 39 1426766 _ at 6330403K07Rik AK018106 1 . 91 60 1447623 _ s _ at Prkcm AV297026 0 . 39 1445081 at A930041102Rik BB335888 1 . 90 1455739 _ at EG245190 BB279146? 0 . 39 1455458 _ x _ at Gmppa AU044197 1 .89 1418355 _ at Nucb2 NM _ 016773 0 . 38 1436182 _ at BG092481 1 . 88 1419517 _ at Cnih3 NM _ 028408 0 . 38 1425484 _ at Tox BB547854 1 .87 1423477 _ at Zici BB361162 0 . 38 1456954 _ at Kcn?? BM119753 1 . 84 1422860 _ at Nts NM 024435 0 . 38 1417434 at Gpd2 NM 010274 1 .83 65 1423367 _ at Wnt7a AK004683 0 . 37 1454708 _ at Ablim1 BG065289 1 . 83 1435479 _ at BM055476 0 . 36 US 9, 816 ,096 B2 71 72 TABLE 10 -continued enriched subtypes and compared to data collected from the total RNA of one whole brain (minus striatum ) ( Table 13 ) . Genes differentially translated between The analysis resulted in detection of several thousand striatonigral and striatopallidal cells. translated mRNAs enriched in striatum relative to whole Probe Symbolbol FCH 5 brain , including all previously - known striatal -enriched GenBank genes: Ppplrlb /Darpp -32 ( S . I. Walaas , P .Greengard , JNeu 1437156 _ at Efcbp1 BB392041 0 . 36 1452135 _ at Gpx6 AV001252 0 . 34 rosci 4 , 84 - 98 (January , 1984 ), ) Ptpn5 / Step ( P . J. Lombroso , 1460623 _ at Skap2 BB753881 0 . 34 J . R . Naegele , E . Sharma, M . Lerner, J Neurosci 13 , 3064 -74 1417577 _ at Trpc3 NM _ 019510 0 . 33 (July , 1993 ), ) Arpp - 19 ( J. A . Girault, A . Horiuchi, E . L . 1439566 _ at Gprin3 BB245373 0 . 33 10 Gustafson , N . L . Rosen , P . Greengard , J Neurosci 10 , 1418639 _ at Sftpc NM _ 011359 0 . 33 1457142 _ at Efcbp1 BB667731 0 . 32 1124 - 33 (Apr , 1990 ) ,) Arpp -21 / RCS ( C . C . Ouimet, H . C . 1439627 _ at Zic1 AVO31691 0 . 31 Hemmings , Jr . , P . Greengard , J Neurosci 9 , 865 - 75 (March , 1454659 _ at Dctd BG069699 0 . 30 1989 ), ) Goal/ Golf ( D . Herve eta! . , J Neurosci 13, 2237 -48 1427300 _ at Lhx8 D49658 0 . 30 (May , 1993 ) , ) Rhes /Rasd2 ( J . D . Falk et al. , J Neurosci Res 1416319 _ at Adk NM 134079 0 . 29 15 57 , 782 - 8 6 ( Sep . 15 , 1999 ), ) Rgs9 ( S . J . Gold , Y . G . Ni, H . 1438292 _ x _ at Adk BB559878 0 . 28 1451191 _ at Crabp2 BC018397 0 . 26 G . Dohlman , E . J . Nestler, J Neurosci 17 , 8024 -37 (Oct . 15 , 1419577 _ at A530089117Rik NM _ 133999 0 . 26 1997 ) , ) Adcy5 ( C . E . Glatt, S . H . Snyder, Nature 361 , 536 - 8 1440177 _ at 9 .63E + 17 BM899529 0 . 26 ( Feb . 11 , 1993) ,) Gng7 ( J . B . Watson et al. , J Neurosci Res 1422586 _ at Eceli NM _ 021306 0 .25 1433787 _ at Nelli AI841091 0 . 23 39 , 108 - 16 ( Sep . 1 , 1994 ) , ) Rasgrp2 ( H . Kawasaki et al . , 1422541 at Ptprm NM _ 008984 0 . 22 20 Proc Natl Acad Sci USA 95 , 13278 - 83 (Oct . 27 , 1998 ), ) 1434458 at Fst BB444134 0 .21 Pdelb (J . W . Polli, R . L . Kincaid , Proc Natl Acad Sci USA 1427519 _ at Adora2a BG311385 0 . 18 89 , 11079 - 83 (Nov . 15 , 1992 ) , ) Pde10a ( K . Fujishige , J . 1433578 _ at Slc10a4 BE824538 0 . 17 1421365 _ at Fst NM _ 008046 0 . 15 Kotera , K . Omori, Eur J Biochem 266 , 1118 - 27 (Dec . , 1424902 at Plxdc1 AF378760 0 . 13 1999 ) , ) Gpr88 ( K . Mizushima et al. , Genornics 69 ,314 - 21 1427038 at Penki M13227 0 . 13 25 (Nov . 1 , 2000 ) ,) Rarb ( W . Krezel, P . Kastner, P . Chambon , 1428547 _ at Nt5e AV273591 1460244 _ at Upb1 NM 133995 0 . 1113 Neuroscience 89, 1291 - 300 ( 1999 ), ) and Strn4 ( F . Castets et 1440148 _ at Gpro A1852874 0 .08 al. , J Cell Biol 134 , 1051- 62 (Aug . , 1996 ) ) as well as the 1460710 _ at Adora2a BB748999 0 . 08 transcription factors Foxpl, Foxp2 ( R . J . Ferland , T . J . 1418950 _ at Drd2 NM _ 010077 0 . 03 Cherry , P . O . Preware , E . E . Morrisey , C . A . Walsh , J Comp 1435211 _ at Ttc12 BB554351 0 . 00 30 Neuro1460 , 266 - 79 (May . 26 , 2003 ) , ) Ebf ( S . Magdaleno et Fold -change values (FCH ) are given relative to D1 ( striatonigral ) cells . al. , PLoS Bio14 , e86 ( 2006 ) ) , and ZJP503 /Nolz ( C . W . Probe = Affymetrix probe identification ; Symbol = official gene symbol. Chang et al. , Proc Natl Acad SciUSA 101 , 2613 - 8 ( Feb . 24 , 2004 ). ) ( Table 13 ). As independent confirmation of these data , mRNA TABLE 11 35 expression patterns were examined for a subset of the candidate MSN -expressed genes in the GENSAT/ Brain Real time PCR analysis of D1 enriched Gene Expression Map ( BGEM ) and Allen Brain Atlas messages: range from replicates . (ABA ) in situ hybridization ( ISH ) databasee (available using Fold enrichment in D1 the hypertext transfer protocol and world wide web at Gene versus D2 cell type 40 ncbi. nlm .nih . gov/ projects /gensat /; available using the Eyal 19 . 85 -21 . 51 hypertext transfer protocol and world wide web at stjudeb Tac1 (known ) 10 . 97 - 11 . 87 gem .org ) . Only those genes for which expression data were Isli 10 . 06 - 10 . 87 available in both gene expression atlases were chosen . The Gng2 5 .91 - 6 .43 Chrm4 ( known ) 5 .02 - 5 .57 data are presented in FIG . 29 . Of the first 100 genes Drdla ( known ) 2 . 96 - 3 . 4 45 appearing in the MSN - enriched dataset, 26 were present in Crym 1 . 57 - 1 .88 both the BGEM and ABA public ISH atlases . Enriched striatal expression is evident for 22 of these genes ( Table 13 ) ; panel a ). ISH data were available in the GENSAT /BGEM and ABA TABLE 12 50 for 16 genes appearing between numbers 1 ,000 to 1 , 100 on Real time PCR analysis of D2- enriched the MSN enriched list. In this case , striatally -enriched messages : range from replicates . expression is apparent for 7 of these genes ( ( Table 13) ; panel b ). Fold enrichment in D2 versus Expression analysis in sagittal sections of genes which Gene D1 cell type were amongst the top 100 ( a ) or 1 ,000 - 1 , 100 ( b ) genes Drd2 (known ) 49. 79 - 51 .55 identified in the study as MSN - enriched , with the rank order Gpro 12 . 30 - 12 . 81 of each gene noted below the gene name. Non -redundant Lhx8 3 .57 - 4 .06 Gpr88 1 . 94 - 2 .13 gene ranking was calculated using the highest - ranked probe Tpc4 1 . 93 - 2 . 25 set corresponding to each gene , with redundant probesets Tpm2 1 . 84 - 1 . 96 60 eliminated . Left panel, in situ hybridization images taken from the Allen Brain Atlas (Allen Brain Atlas, Internet . Seattle (WA ) : Allen Institute for Brain Science . © 2006 . ; 4 ) Large Scale Validation of BACarray Profiling of Stria Available using the hypertext transfer protocol on the world tonigral and Striatopallidan MSNs wide web at brain -map .org ; E . S . Lein et al. , Nature 445 , Large - scale validation of the data using publicly available 65 168 - 76 ( Jan . 11 , 2007 ) ) ; right panel , in situ hybridization gene expression databases was carried out. Data were pooled images taken from the Brain Gene Expression Map ( BGEM ) from D1 and D2 BACarray experiments to representMSN - database (available using the hypertext transfer protocol on US 9 ,816 , 096 B2 73 74 the world wide web at stjudebgem . org /) ( S . Magdaleno et TABLE 13 - continued al. , PLoS Biol 4 , e86 ( Apr. , 2006 ) . Allen Brain Atlas images all correspond to adult brain ; BGEM images all correspond Genes differentially translated between medium to adult brain except for the following , for which the oldest spiny neurons (MSNs ) and the rest of the brain . available data were postnatal day 7 (P7 ) : Drd2 , Ppplrlb , 5 Probe Symbol GenBank FCH Dlx6 , Gdnf, Bcl11b , Foxgl, Limd2 , Femlb , Dynl11 , Atbfi , 1429229 _ s _ at 4930534B04Rik BE980134 15 . 28 Foxo3a , Dnalc4 , Mtmr7 , Dnmt3a. 1454762 _ at Nox1 BM932447 15 . 26 1419390 _ at Pde10a BQ180352 15 . 22 TABLE 13 1435366 _ at D430042009Rik BB486367 15 . 19 10 1425756 _ at Rab40b AF425643 15 . 18 Genes differentially translated between medium 1418744 _ s _ at Tesc NM _ 021344 14 .99 spiny neurons (MSNs ) and the rest of the brain . 1416627 _ at Spint1 NM 016907 14 . 90 1436650 _ at Filip1 AV241894 14 . 84 Probe Symbol GenBank FCH 1460136 _ at AW047481 A1462839 14 . 68 1435808 _ at A230051G13Rik BB174377 14 . 53 1420437 _ at Indo NM _ 008324 198 .43 1421140 _ a _ at Foxpl BG962849 14 . 38 1427343 _ at Rasd2 BC026377 149 . 45 1437698 _ at Myo5b AV370579 14 . 36 1457132 _ at BF456117 133 .95 1416456 _ a _ at Chia BC011134 14 . 31 1458342 _ at Tmem90a BB313069 108 .01 1442166 _ at Cpne5 BB273427 14 . 21 1418950 _ at Drd2 NM _ 010077 85 . 64 1456051 at Drdla BB282271 13. 84 1438355 _ at Tmem90a A1414870 79 .94 1421365 _ at Fst NM _ 008046 13 . 82 1441368 at BB102769 71 . 65 1420496 _ at F12 NM __ 021489 13 .79 1426814 _ at AU024582 BM248309 57 . 27 20 1436092 _ at BB336256 13. 75 1418782 _ at Rxrg NM _ 009107 56 .75 1448327 at Actn2 NM _ 033268 13 .69 1423171 at Gpr88 BE947345 52 . 53 1429228 _ at 4930534B04Rik BE980134 13 .65 1457143 _ at LOC665139 BB322292 50 . 59 1419826 _ at Kif17 AW492270 13 .61 1426438 _ at Ddx3y AA210261 49 . 33 1436734 _ at E130309F12Rik BB523550 13 . 60 1419696 _ at Cd4 NM _ 013488 45 . 51 1421353 at Pde7b NM 013875 13 . 54 1458499 _ at Pde10a AW123977 41 .50 25 1425833 _ a _ at H??? AF326551 13 . 53 1427344 _ s _ at Rasd2 BC026377 34 .92 1454656 _ at Spatal3 AV271736 13 . 50 1450213 at Pde7b NM 013875 33 .08 1455298 _ at Id4 BB306828 13. 39 1455190 _ at Gng7 BM114283 31. 37 1425363 at B4galnt1 BC022180 13 . 23 1429285 _ at Serpina9 AK009343 29 .48 1439854 _ at Hrk BQ175572 13 . 22 1454906 _ at Rarb BB266455 28 . 73 1434008 _ at Scn4b BE993937 13 . 11 1443287 _ at Gm1337 BB555669 28 .56 30 1417812a at Lamb 3 NM _ 008484 13 . 10 1428642 _ at Slc35d3 AK018094 28 . 48 1435221 _ _ at Foxpl BM220880 13. 06 1417680 _ at Kcna5 NM _ 008419 28 .33 1429617 _ at Cyld BM119209 12. 96 1451331 _ at Ppplrlb BC026568 28 . 25 1418357 _ at Foxgl NM _ 008241 12 . 81 1442558 at Ptprn2 BB180191 27 .93 1436532 _ at Dcamk13 BB326709 12 . 78 1454867 _ at Mn1 BB234631 27 . 26 1441468 _ at BB326028 12 . 77 1437750 _ at Tmem158 BI133257 26 . 54 1447861 _ _ x _ at Mrgl AV329643 12 . 66 1457919 _ at DO30054H15Rik BB446049 25 . 08 1447766 _ x _ at Limd2 AV003249 12 .63 1427807 _ at ENSMUSG00000060559 BC017159 24 .97 1454877 _ at Sertad4 BQ174721 12 .63 1436566 _ at Rab40b AV364488 24 .65 1418847 _ at Arg2 NM 009705 12 . 57 1456818 _ at Stk32a BB279083 24 .52 1422720 _ at Isl1 BQ176915 12 . 56 1452507 _ at Dlx6 AF022078 24 . 32 1417704 _ a _ at Arhgap6 NM _ 009707 12 . 45 1457651 x _ at Rem2 BB270375 24 .03 1455361 at Dgkb AW493391 12 . 28 1428781 _ at Dmkn B1452905 23. 79 40 1432073 _ at 1700113122Rik AK007198 12 . 27 1460710 _ at Adora2a BB748999 23 . 75 1418743 _ a _ at Tesc NM _ 021344 12 . 18 1455629 _ at Drdla BE957273 23. 51 1444734 _ at A330001L22 Rik BB183877 12 . 17 1422079 _ at Prkch NM _ 008856 22 . 36 1433815 at Jakmip1 AV290082 12 . 08 1435296 _ at Adra2c AV349563 21. 79 1452217 _ at Ahnak BE570050 12 .02 1440148 at Gpr6 A1852874 20 . 60 1452135 _ at G??6 AV001252 11. 98 1437935 _ at 4930486G11 Rik BB821151 19 .59 45 1460325 _ at Pum1 BB837171 11 .96 1427523 _ at Six3 A1509029 19 . 13 1439618 _ at Pde10a A1448308 11 . 94 1419389 _ at Pde10a BQ180352 19 . 09 1434521 _ at Rfxdc2 BB148972 11. 92 1454581 _ _ at 5330425B07Rik AK019908 19 .03 1435285 _ at Mpped2 BB731805 11. 92 1440166 _ x _ at Htrld BB378627 18 . 87 1416266 _ at Pdyn AF026537 11 . 84 1426815 _ s _ at AU024582 BM248309 18 .68 1422230 _ s _ at Cyp2a5 NM _ 007812 11 . 82 1419080 _ at Gdnf NM _ 010275 17 .98 50 142270622706 __ _ at Tmepai AV370981 11 .81 1435211 _ _ at Ttc12 BB554351 17 . 93 1448468 _ a _ at Kcnab1 AF033003 11. 74 1438022 _ at Rab11fip3 BO266518 17 . 69 1434917 at Cobl BO173923 11. 68 1445539 _ at BE687857 17 .64 1418881 _ _ at Efcbp2 NM _ 054095 11 . 68 1449420 _ at Pdelb NM _ 008800 17 . 44 1433743 _ at Dach1 BG075820 11 . 67 1427683 _ at Egr2 X06746 16 .79 1441733 _ s _ at Nup153 C88147 11. 66 1456640 _ at Sh3rf2 AW910872 16 .61 1442077 at Htrlb BB197581 11. 66 1456280 _ at Clspn BG067086 16 . 53 1433566 _ at Rasl10b BB381618 11 . 54 1451236 _ at Rerg BC026463 16 . 46 1423117 _ at Pum1 BB837171 11. 50 1460262 a at 1700037H04Rik NM 026091 16 . 39 1426439 _ at Ddx3y AA210261 11 . 48 1460038 _ at Pou3f1 BG065255 16 . 23 1436216 _ s _ at 2610204M08Rik BM234799 11. 46 1443694 _ at Rgs20 BB794177 16 . 06 1431751 _ a _ at Mpped2 AKO12553 11 . 40 1417804 _ at Rasgrp2 NM __ 011242 16 .01 1437865 _ at Spatal3 AW546433 11 . 40 1451280 _ at Arpp21 BB159263 15 . 99 60 1418691 _ at Rgs9 NM _ 011268 11 . 39 1450339 _ a _ at Bcliib NM _ 021399 15 .73 1442058 _ S _ at Psmc3ip BE687992 11. 38 1449977 _ at Egr4 NM _ 020596 15 .61 1419033 _ at 2610018G03Rik AW556821 11. 36 1457424 _ at Eyal BB760085 15 .60 1455564 _ at Bor BQ176236 11 . 36 1441000 _ at EG237749 BB076832 15 .52 1455156 _ at Strn BG519214 11. 35 1454043 _ a _ at Kcnabi AK015412 15 . 48 1434664 _ at 2410129H14Rik BI153133 11 . 33 1438841 _ s _ at Arg2 AV002218 15 .44 65 1415953 _ s _ at Mark2 BI686265 11. 31 1417210 _ at Eif2s3y NM _ 012011 15 . 28 1417129 _ a _ at Mrgl U68384 11. 26 US 9, 816 ,096 B2 75 76 TABLE 13 -continued TABLE 13 - continued Genes differentially translated between medium Genes differentially translated between medium spiny neurons (MSNs ) and the rest of the brain . spiny neurons (MSNs ) and the rest of the brain . Probe Symbol GenBank FCH 5 Probe Symbol GenBank FCH 1436275 _ at Kcnip2 AW490636 11 . 23 1453799 _ at 9430038101Rik AKO20460 8 . 31 1456515 _ s _ at Tcf15 AV044715 11. 15 1438224 _ at Zswim5 BE572265 8 . 28 1453222 _ at 4833427B12Rik BE863648 11 .04 1430864 _ at Ttl19 AKO15740 8 .25 1425870 a at Kcnip2 AF439339 10 .91 1451008 _ at St8sia3 BB360510 8 . 25 1420666 _ at Doc2b BM117900 10 . 75 10 1453330 _ at 0610010D24Rik AK002458 8 . 24 1459941 _ _ at Asph BB096900 10 . 73 1420872 _ at Gucy1b3 BF472806 8 . 22 1448913 at Smarcd1 NM 031842 10 .71 1444383 at 1110014J01 Rik AV347845 8 . 21 1440741 at Htrld BB829587 10 . 68 1424132 at Hras1 BC011083 8 . 20 1457038 _ at Frem2 BM201912 10 .66 1420695 at Dach1 BB522228 8 . 13 1458406 _ at A1429294 BG144063 10 . 63 1438266 _ at Adamts15 BB764453 8 .09 1430686 _ at 4833418 NO2Rik AKO14719 10 . 62 15 1456054 _ a _ at Pum1 BB314559 8 .08 1446877 _ at C230014012Rik BB381622 10 .52 1422157 _ a _ at Itgb1bp1 NM 008403 8 .06 1441928 _ x _ at Ell BB139475 10 . 44 1420961 _ a _ at Ivnslabp NM _ 028582 8 . 05 1416350 _ at Klf16 NM _ 078477 10 . 40 1424899 _ at Nmnat3 BC005737 7 .97 1444978 _ at BM223267 10 . 36 1424409 _ at Cldn23 BC019534 7 .94 1417356 _ at Peg3 AB003040 10 . 34 1435071 _ at Zfyvel AV327165 7 . 93 1431386 _ s _ at Mbtps1 AK002809 10 . 32 1437353 _ at Setdlb BB494182 7 . 87 1421943 _ at Tgfa M92420 10 . 27 1456830 _ at Ppp1r2 BB542221 7 . 87 1435553 _ at Pdzd2 AV376136 10 . 17 1445606 _ a _ at 2900009 JO6Rik BB663189 7 . 87 1427931 _ s _ at Pdxk BG063905 10 . 16 1435227 _ at BM117007 7 . 85 1427682a at Egr2 X06746 10 .09 1446455 _ at BB520649 7 . 85 1444681 _ at Erc2 BB749686 10 .07 1443676 _ at Slc4a4 AV339935 7 . 83 1422153 _ a _ at Asb11 NM 026853 10 .00 1429316 at Rasgefla AK018120 7 .82 1439566 _ at Gprin3 BB245373 9 . 99 25 1438417 _ at Pwwp2 BB809239 7 . 82 1420146 _ at Tfblm A1429207 9 . 98 1434996 _ at Slc25a16 AV316233 7 .79 1449957 _ at Ptprv NM _ 007955 9 . 95 1422621 at Ranbp2 BM507707 7 .79 1426241 _ a _ at Scmh1 AB030906 9 . 94 1452176 _ at Nup153 BB292874 NNNNNNN7 . 78 1424208 _ at Ptger4 BC011193 9 . 86 1437460 _ x _ at Rin1 BB264363 7 .78 1450026 _ a _ at B3gnt2 AV306734 9 . 85 1447642 _ x __ at Dmwd AV354897 7 . 70 1436738 _ at Pifi AV094878 9 . 75 30 1417341 a at Ppp1r2 NM _ 025800 7 .69 1434248 _ at Prkch BM243756 9 . 75 1431349 _ at Hnrpab AKO13709 7 .65 1422068 _ at Pou3fi NM _ 011141 9 .75 1438441 _ at Id4 AI323288 7 . 64 1448374 _ at Med28 AK005130 9 .72 1416242 at Klhl13 NM _ 026167 7 .64 1456760 _ at Centg3 BB342676 9 . 70 1441963 _ at Ubox5 BB427703 7 .64 1422813 _ at Cacng1 NM _ 007582 9 .69 1422678 _ at Dgat2 AK002443 7 .60 1449425 _ at Wnt2 BC026373 9 .64 35 1447359 _ at Zfp575 AI326876 7 . 58 1438799 _ at Dlxbos1 AV338180 9 . 63 1450183 _ a _ at Sh2b3 NM _ 008507 7 .58 1456283 _ at tolonciniilialiinilli111:11111iidilisisalelaalipimlNetol AV346211 9 .57 1451517 _ at Rhobtb2 AF420001 7 .55 1425351 at Srxn1 BC011325 9 . 57 1437017 _ at A1480653 BB046727 7 . 54 1424576 _ s _ at Cyp2c44 BC025819 9 . 56 1455296 _ at Adcy5 BE952286 7 . 52 1423544 _ at Ptpn5 BB188812 9 .53 1422779 _ at Smpd3 NM _ 021491 7 .49 1433923 _ at Krt77 AV230775 9 .53 1434531 at Mgat5b BB400317 7 . 46 1430640 _ a __ at Prkar2b BI695530 9 . 52 40 1450072 _ at Ash11 BG694892 7 . 45 1427779 _ a _ at Cd4 U75219 9 . 51 1422335 _ at Adra2c NM _ 007418 7 .45 1452966 _ at 9130430L19 Rik AKO20296 9 . 46 1417062 _ at Armc10 NM 026034 7 .45 1425130 _ a _ at Ptpn5 U28216 9 .41 1426454 _ at Arhgdib AK002516 7 .38 1440711 _ at C630001618Rik BB426320 9 . 37 1443073 at LOC545681 BB355954 7 .38 1431936 _ a _ at Neu2 AK009828 9 . 36 1458407 _ s _ at A1429294 BG144063 1434025 _ at Klf5 BG069607 9 . 33 45 1453323 _ at 2900079G21 Rik AKO13806 7 .34 1441867 _ x _ at 4930534B04Rik A1480494 9 . 30 1438664 _ at Prkar2b BB216074 7 .27 1427912 at Cbr3 AK003232 9 . 27 1428839 _ at Wdr53 AK005105 - .27 1436002 _ at BB484128 9 . 23 1442708 _ at BB504418 7 .26 1449502 _ at Dazl NM _ 010021 9 . 21 1418467 _ at Smarcd3 NM _ 025891 7 . 25 1430612 _ at 1810033B17Rik BB533148 9 . 19 1455401 _ at Camkk2 AW061083 7 . 24 1455242 _ at Foxp1 BM220880 9 . 18 50 1434732 _ x _ at Tomm7 AV044898 7 . 24 1441105 _ at BE980314 9 . 16 1429618 _ at Cyld BM _ 119209 7 .23 1447017 at BE956696 9 .03 1428860 _ at 4930572J05 Rik AKO19809 7 .22 1435334 _ at Ttc7 BG866904 8 . 97 1453264 _ at Marveld3 AK007346 7 .22 1436998 _ at Ankrd43 BB428991 8 .96 1421142 _ s _ at Foxpl BG962849 7 .22 1454086 _ a _ at Lmo2 AKO13416 8 . 87 1418714 _ at Dusp8 NM 008748 7 . 20 1419542 at Dazl NM _ 010021 8 . 82 55 1452077 _ at Ddx3y AA210261 7 . 19 1442234 _ at Chst2 BB770422 8 . 72 1428370 _ at 1500011 B03Rik AKO18264 7 . 19 1449331 _ _ a _ at Rapsn NM 009023 8 .68 1454832 _ at Phactr1 BG228702 7 . 19 1442021 _ _ at Mppel BB502545 8 . 65 1434153 _ at Shb BI408715 7 . 18 1429201 at Cyld AKO13508 8 .55 1456475 _ s _ at Prkar2b BB216074 7 . 15 1440435 _ at Ky BB126077 8 . 55 1422722 _ at 1700001K19 Rik NM _ 025488 7 . 14 1441350 _ at Fgf3 AV302620 8 .53 1434495 _ at Patzl BM208058 7 . 14 1434519 at Ddah1 AW556888 8 . 53 60 1423532 _ at Rnf44 A1850285 7 . 12 1452298 _ a _ at Myo5b AW546331 8 . 51 1437091 at Accn4 AV323885 7 . 11 1422407 _ s _ at Hras1 NM _ 008284 8 . 45 1418017 _ at Pum2 BI689507 7 . 10 1416996 _ at Tbc1d8 BC005421 8 . 43 1423049 _ a _ at Tpm1 AK002271 7 . 10 1428873 _ a _ at 4121402D02 Rik AW495537 8 . 37 1455080 _ at Ppp1r16b BB375209 7 . 10 1422448 at Tf2 NM 009363 8 . 36 1416239 _ at Ass1 NM 007494 7 . 10 1443036 _ at Zfp804a BG073535 8 . 35 65 1428338 _ at Spata2L AKO19166 7 . 07 1417522 _ at Fbxo32 AF441120 8 . 32 1425104 _ at Kctd1 BC022615 7 .06 US 9, 816 ,096 B2 77 78 TABLE 13 - continued TABLE 13 - continued Genes differentially translated between medium Genes differentially translated between medium spiny neurons (MSNs ) and the rest of the brain . spiny neurons (MSNs ) and the rest of the brain . Probe Symbol GenBank FCH Probe Symbol GenBank FCH 1433627 _ at Sec23ip AW546839 7 .02 1439576 _ at BB325333 6 . 34 1455085 _ at 1700086L19 Rik BI526033 7 .02 1428777 _ at Spred1 AK017680 6 . 34 1416804 _ at Ehbp111 NM _ 053252 7 .00 1430413 _ at Tmem29 AKO18228 6 . 34 1452729 at Dpm3 BE446919 7 .00 1422293 _ a _ at Kctd1 NM _ 134112 6 .33 1419234 _ at Helb BG070273 7 .00 10 1423488 _ at Mmd BC021914 6 . 33 1449261 _ _ at Pbx2 NM _ 017463 6 . 99 1436645 _ a _ at Cnot4 BB066603 6 . 32 1456336 _ at A330102K23Rik BB182912 6 .99 1445404 at Kif27 BB054454 6 . 32 1416983 _ s _ at Foxol A1462296 6 .99 1428547 _ at Nt5e AV273591 6 . 32 1442220 _ at BB313387 6 . 97 1454752 _ at Rbm24 AV307961 6 . 29 1455961 _ at AV174022 6 .96 1452092 _ at 4631426J05 Rik AK019474 6 . 28 1460567 _ at Rfxdc2 BB148972 6 .94 15 1447830 _ s _ at Rgs2 BB034265 6 . 27 1437579 _ at Nek2 C77054 6 . 94 1455447 _ at D430019H16Rik BM116882 6 .26 1452913 _ at Pcp411 AV337888 6 . 93 1452904 _ at 1700026L06Rik BQ175881 6 . 26 1441223 at 3 -Mar BB260801 6 .93 1444363 _ at BB269445 6 . 26 1435504 _ at Clip4 BM217861 6 .93 1437839 _ x _ at Mrpl11 AV209741 6 . 25 1434077 _ at Wdr37 AV222037 6 . 91 1445385 _ at BB551855 6 . 25 1452879 _ at Synpo2 A1848603 6 .90 1419247 _ at Rgs2 AF215668 6 .24 1460285 _ at Itga9 NM _ 133721 6 . 90 20 1416886 _ at Cid NM _ 020558 6 .21 1424796 _ at 1700054NO8Rik BC024705 6 . 89 1419378 _ a _ at Fxyd2 NM _ 052823 6 . 20 1435125 at BB120497 BB303627 6 . 87 1450366 _ at Hrk NM _ 007545 6 . 19 1433962 _ at 6720458F09Rik BB131965 6 . 86 1428495 at 2410003K15 Rik BF300229 6 . 19 1429098 _ s _ at Nhej1 AK006481 6 .85 1439844 _ at 8430426J06Rik BE628126 6 . 18 1452411 _ at Lrc1 BG966295 6 .85 1455345 _ at Phf15 B1663145 6 . 16 1422159 _ at Ppef2 BC027049 6 . 83 25 1449304 _ at 2310061J03Rik NM _ 133677 6 . 16 1452166 _ a __ at Krt10 AK014360 6 . 83 1435564 _ at C230078M08Rik BB547893 6 . 16 1416981 _ at Foxol A1462296 6 . 82 1451819 _ at Zswimo BC021311 6 . 16 1437092 _ at Clip4 AV042829 6 .81 1449141 at Fblim1 BG070068 6 . 15 1430306 _ a _ at Atpovic2 AK004157 6 . 80 1448860 _ at Rem2 NM 080726 6 . 15 1421442 _ at Flt4 NM _ 008029 6 .77 1450835 _ a _ at Gfra4 NM _ 020014 6 . 15 1435649 at Nexn BM225804 6 .75 30 1435096 _ at Ric8b BB667093 6 . 15 1455683 _ a _ at Tbc1d8 BB451404 6 .73 1454901 _ _ at Ypel2 BG069663 6 . 15 1434649 _ at Ccm2 B1903794 6 . 72 1423270 _ at Nedd41 BB663717 6 . 14 1455804 __ x _ at Oxct1 AV213379 6 .71 1452202 at Pde2a BG069616 6 . 12 1423344 _ at Epor AKO10968 6 .69 1442754 _ at C030013G03Rik BE692283 6 . 10 1460555 _ at 6330500D04Rik BM242294 6 .68 1449863 _ a _ at Dlx5 NM __ 010056 6 .09 1436799 _ at Rp123a BB386177 6 .67 35 . 1422134 _ at Fosb NM 008036 6 .09 1426687 _ at Map3k3 BG175594 6 .66 1430222 _ at 9130007G19Rik BB538672 6 .09 1434032 _ at Centg3 BQ175381 6 .65 1434648 _ a _ at Ccm2 B1903794 6 . 09 1423530 _ at Stk32c BB320288 6 .63 1428139 at Tmem180 AKO15988 6 . 08 1443543 _ at BE980616 6 .63 1439947 _ at Cypllal C87524 6 .08 1447100 _ s _ at C80506 6 .62 1421141 _ _ a _ at Foxpl BG962849 6 . 08 1437842 at Plcxd1 BB311508 6 . 59 1415976 a at Carhsp1 AU080787 6 . 07 1420534 _ at Gucyla3 AK004815 6 .59 40 1433988 _ s _ at C230098021Rik BG075755 6 .06 1455301 _ at Wipf3 BG064092 6 . 59 1422705 _ at Tmepai AV370981 6 .06 1429637 _ at 2210419108Rik AK008987 6 . 57 1443334 _ at D430042009Rik BB487579 6 .06 1442051 _ at Hist2h3c1 BE691662 6 .54 1434909 _ at Rragd BF462770 6 . 04 1434458 at Fst BB444134 6 .54 1456930 _ at Camsap1 BE989461 6 .03 1435605 at Actr3b BB125424 6 .53 1416605 _ at Nola2 BC024944 6 .03 1422034 _ a _ at Palm NM _ 023128 6 . 53 45 1417090 _ at Rcnl NM 009037 6 .02 1419742 at 1700037H04Rik NM 026091 6 .52 1460686 _ at Cntd1 BC006866 6 .02 1438171 _ x _ at Mett19 BB056666 6 . 52 1430190 _ at 1700041 CO2Rik AK006668 6 .02 1448807 _ at Hrh3 NM 133849 6 .50 1430616 _ at 4930528A17Rik BB185833 6 .01 1437097 _ at Zbed3 BB703866 6 .49 1419248 _ at Rgs2 AF215668 5 . 99 1424759 _ at Arrdc4 BC017528 6 . 49 1417524 _ at Cnih2 NM _ 009920 5 . 97 1438210 _ at Gpr149 BB126999 6 .48 50 1453260 _ a _ at Ppp2r2a AKO10380 5 . 95 1441340 _ at Cep68 BF018298 6 .47 1443612 _ at Tmem16c AW123199 5 . 95 1443887 _ at Ankrd13c BE132758 6 .47 1424248 at Arpp21 BB159263 5 .93 1422608 at Arpp19 BE648432 6 .45 1449843 at St8sia2 BG071333 5 .93 1452917 _ at Rfc5 AK011489 6 .43 1420391 _ at Pard3 NM _ 033620 5 . 93 1416982 _ at Foxo1 A1462296 6 .43 1434115 _ at Cdh13 BQ176681 5. 92 1418894 _ s _ at Pbx2 NM _ 017463 6 .42 55 1457052 _ at AW536275 BG064867 5 .92 1449158 _ at Kenk2 NM _ 010607 6 .42 1456838 at BC072620 BE995608 5 . 92 1448978 _ at Ngef NM 019867 6 .41 1436538 at Ankrd37 AVO84342 5 .91 1427196 _ at Wnk4 BG074348 6 .41 1438226 _ at AU022252 AV349089 5 .90 1424507 _ at Rin1 BC011277 6 .40 1422208 _ a _ at Gnb 5 BC016135 5 .89 1441089 _ at Eif2c1 BB205493 6 . 40 1452368 _ at Bcr A1853148 5 .88 1427293 _ aat Auts2 AKO12475 6 . 39 1416488 _ at Cong2 U95826 uuuuuuuuuuu5 . 87 1457195 _ at Plekhm1 BB206527 6 . 38 60 1435222 _ at Foxpl BM220880 5 . 87 1436930 _ x _ at Hmbs BB000512 6 . 37 1432579 _ at Rsh12b AA544511 5 . 85 1434359 at 6330500DO4Rik AV329070 6 . 37 1417658 _ at Tbrg4 NM _ 134011 5 . 85 1420444 _ at Slc22a3 NM _ 011395 6 . 37 1422923 at Fgf3 NM 008007 5 . 85 1417440 _ at LOC675933 NM _ 033566 6 . 37 1421037 _ at Npas2 BG070037 5 .85 1425503 _ at Gent2 AB037596 6 . 36 1431711 _ a at 9030409G11 Rik AKO18497 5 .85 1438784 _ at Bcliib BB329234 6 . 34 65 1431242 _ at 6330575P09Rik BE995561 5 . 85 1460044 _ at Onecut2 BB389395 6 . 34 1452911 _ _ at Spred1 AK017680 5 . 84 US 9, 816 ,096 B2 79 80 TABLE 13 -continued TABLE 13 - continued Genes differentially translated between medium Genes differentially translated between medium spiny neurons (MSNs ) and the rest of the brain . spiny neurons (MSNs ) and the rest of the brain . Probe Symbol GenBank FCH Probe Symbol GenBank FCH 1458133 _ at BB360008 5 .81 1454256 _ s _ at 1700113122Rik AK004415 5 .22 1438019 _ at Ippk BM196347 5 . 81 1427954 _ at BC048403 BC028785 5 . 21 1417003 _ at 0610012G03Rik BC021536 5 . 80 1450227 _ at Ankrd6 BM199504 5 .21 1448231 _ at Fkbp5 U16959 5 . 80 1454309 _ at 2810002NO1 Rik BB646622 5 . 20 1428074 _ at Tmem158 BE981853 5 .80 10 1435941 _ at Rhbd13 BM118307 5 . 20 1456401 _ at Cacnb2 BB078175 5 . 79 1450723 _ at Isli BQ176915 5 . 19 1423376 a at Dok4 AV341904 5 .77 1419006 _ s _ at Peli2 NM 033602 5 . 18 1443365 at Htr4 BB431008 5 . 76 1434301 _ at D330050123Rik BE303700 5 . 18 1416087 _ at Apls1 NM _ 007457 5 .76 1423442 _ a _ at Fbxw2 BE854125 5 . 18 1429104 _ at Limd2 AKO12581 5 .76 1434828 _ at B430201A12Rik BM200103 5 . 18 1435218 _ at Rasgefla BI134758 5 .75 15 1431717 at 3526401B18Rik AK014386 5 . 18 1454742 _ at Rasgeflb BB003229 5 . 75 1439028 _ at Ufml A1415161 5 . 17 1437948 _ x _ at Eif3ship BB443362 5 .75 1444615 _ x _ at Runxit1 AV327778 5 . 17 1447640 _ s _ at Pbx3 BB554874 1434775 _ at Pard3 AW543460 5 . 17 1460718 _ s _ at Mtch1 AF192558 5 . 74 1437980 _ at 9130230NO9Rik BB814947 5 . 16 1453972 _ x _ at Pparbp AK005207 5 . 74 1455923 at Kctd8 BB352927 5 . 16 1430520 _ at Cpne8 AW548480 5 . 71 1455200 _ at Pak6 BB818370 5 . 15 1418015 _ at Pum2 BI689507 5 .70 20 1416479 a at Tmem14c NM _ 025387 5 . 15 1455701 _ at Snx26 B1965517 5 .67 1452964 _ at Ttll11 AK016577 5 . 15 1436160 _ at Krt26 BB150142 5 .67 1422440 _ at Cdk4 NM _ 009870 5 . 15 1458025 at Ttc7 BB519333 5 . 66 1420694 _ a _ at Dach1 BB522228 5 . 15 1427005 _ at Plk2 BM234765 5 .66 1456197 _ x _ at BB342782 5 . 14 1434682 _ at Zfp770 AV347367 5 .64 1418592 _ at Dnaja4 NM 021422 5 . 14 1439291 _ at BB354792 5 .64 25 1421234 _ at Tcfi NM 009327 5 . 13 1438680 _ at Auts2 BB429147 5 .63 1425753 _ a _ at Ung BC004037 5 . 13 1443552 _ at E230008N13Rik BM932485 5 .63 1426964 _ at 3110003A17Rik AK013984 5 . 13 1453089 _ at 3110079015Rik AA259365 5 .62 1420778 _ at Tas1r3 NM _ 031872 5 . 12 1440501 _ _ at AU041480 BG072095 5 . 62 1433610 _ at AA986860 BB522283 5 . 12 1440940 _ at Cacnb1 BE949510 5 .62 1422807 _ at Arf5 NM _ 007480 5 . 11 1446840 _ at Thrap1 BQ032894 5 .62 30 1447559 _ at A1834762 A1834762 5 . 10 1460327 _ at Gpr88 BE947345 5 .57 1434014 _ at Atg4c BB291836 5 . 10 1426230 _ at Sphk2 AK016616 5 .55 1433642 _ at Arl15 BB384173 5 . 09 1419130 _ at Deadcl NM _ 025748 5 .54 1426573 _ at Me2 BM235734 5 .08 1434967 _ at Zswim BM938007 5 . 53 1422165 _ at Pou3f4 X66603 5 .08 1432782 at 4933435C09Rik AKO17064 5 .52 1431186 _ at Dlg5 BF140264 5 .08 1441967 _ at Pddc11 BB467791 5 .51 35 1429422 _ at 4933412E12Rik AKO16788 5 .08 1433468 _ at 6430527G18Rik BB770958 5 . 51 1453309 _ at 9330179D12 Rik BB749938 5 .08 1435741 at Pde8b BB312125 5 .49 1457512 at BB167650 5 . 08 1437400 _ at Nedd41 BB309512 5 .48 1435615 _ at Zfp365 BB277790 5 . 08 1460206 _ at Grasp NM _ 019518 5 .47 1437378 _ x _ at Scarb1 BB224405 5 .07 1433767 _ at 1110018G07Rik AV257687 5 . 45 1429124 _ s _ at Rexol BQ178367 5 . 07 1420918 _ at Sgk3 BB768208 5 .45 1455153 _ at Zfp236 BQ177220 5 .07 1438189 _ s _ at Rai16 BB492312 5 .44 40 1430617 _ at Oip5 BB238604 5 . 06 1433507 _ a _ at Hmgn2 BE553881 5 .42 1427519 _ at Adora2a BG311385 5 .06 1450930 _ at Hpca AK002992 5 .42 1436142 _ at 3526401B18Rik BM218877 5 . 06 1418373 at Pgam2 NM 018870 5 . 40 1438068 _ at BB251859 5 . 04 1427278 _ at Clip4 AK017914 5 . 40 1429678 at 5730508B09Rik AKO17758 5 .04 1430013 _ at Git1 BE993118 5 .39 1455022 _ at Strn BG070684 5 .03 1437586 _ at Cnot4 BB756908 5 . 38 45 1448684 _ at Ppp1r2 NM _ 025800 5 .03 1439821 at Lrp2bp BB075157 5 .38 1423639 _ at Hrh2 AKO20259 5 .02 1453268 _ at Thg11 AV224102 5 . 37 1451998 _ at Tasp1 BC024597 5 . 02 1451483 _ s _ at 1700054NO8Rik BCO24705 5 . 35 1460166 _ at Rit2 BB271919 5 . 02 1436725 _ at E130306D19Rik BM226118 5 . 34 1451604 _ a _ at Acvrli BC014291 5 . 02 1425123 _ at BC025816 BC025816 5 . 34 1450418 _ a _ at Yipf4 NM _ 026417 5 .01 1442434 _ at D8Ertd82e BM195829 5 . 32 50 1453000 _ at Camsap111 AK005444 5 . 01 1444379 _ at Pwwp2 BB318408 5 . 31 1438665 _ at Smpd3 BF456582 5 .01 1416558 at Melk NM 010790 5 . 30 1432184 _ a _ at 2610204MO8Rik AK011884 5 .01 1439855 _ at Tmtc1 AV341449 5 . 30 1428095 _ a at Tmem24 AK004920 5 .01 1427045 _ at Synpo A1849322 5 . 30 1427745 __ x _ at Promp5 X63005 5 .01 1450480 _ a _ at Gprko AF040749 5 . 30 1423802 _ at Camkv BC017634 5 .00 1427015 _ at C230021 PO8Rik BI732921 5 . 30 55 1417897 _ at Brms1 NM _ 134155 5 .00 1417829 _ a _ at Rab15 NM _ 134050 5 . 29 1458967 _ at BM939341 5 .00 1457632 _ s _ at Mrg1 BB207647 5 .29 1420950 _ at Znrfi BB026596 4 .99 1453040 _ at 2810402A17Rik AK013345 5 .28 1418783 _ at Trpm5 AF228681 4 . 99 1445459 _ at Sstr5 BB023293 5 . 27 1438613 _ at Kcna4 BB131475 4 .98 1455670 _ at BQ175884 5 . 27 1433996 _ at Suv39h2 BB296443 4 . 97 1424475 _ at Camkk2 B1157430 5 . 26 1435423 _ x _ at 4933433P14Rik AV281088 4 . 96 1424020 _ at Arlóip6 BB837198 5 . 26 60 1417082 _ at Anp32b NM _ 130889 4 . 96 1444522 _ at BM942961 5 . 26 1441883 _ at 0610010012Rik AV092359 4 . 95 1434124 __ x _ at 2400001 EO8Rik AV100071 5 .26 1456225 _ x __ at Trib3 BB508622 4 .95 1436100 _ at Sh2d5 AV347995 5 .25 1434154 _ at Kctd13 BQ177107 4 .95 1437453 _ s _ Pcsko AV010795 5 .24 1423489 _ at Mmd BC021914 4 .94 1431665 _ a _ at Timm8b AK004190 5 .23 1452476 _ at Cacnb2 W41214 4 .94 1420752 _ at Dtx3 NM 030714 5 .23 65 1447844 _ at AV077281 4 . 93 1453165 _ at Mettl4 BB701076 5 .22 1433668 _ at Pnrc1 BI410130 4 . 93 US 9, 816 ,096 B2 82 TABLE 13 -continued TABLE 13 - continued Genes differentially translated between medium Genes differentially translated between medium spiny neurons (MSNs ) and the rest of the brain . spiny neurons (MSNs ) and the rest of the brain . Probe Symbol GenBank FCH 5 Probe Symbol GenBank FCH 1416398 _ at Mesdc1 BB474887 4 .93 1420533 _ at Gucyla3 AK004815 4 . 61 1423260 _ at Id4 BB121406 4 . 92 1418169 _ at Zcchc14 BB223737 4 .61 1440108 _ at Foxp2 BM964154 4 .92 1443542 _ at BB767151 4 . 60 1430439 at Mctp1 AK013379 4 .91 1428676 _ at Tmprsso AK004939 4 .60 1440426 _ at Nfatc2 BB427970 4 .91 10 1437146 _ x _ at Coro7 AVO25980 4 .60 1437181 _ _ at Peli2 BM121149 4 .89 1428535 _ at 9430020K01 Rik AK004276 4 .59 1422474 at Pde4b BM246564 4 .89 1418173 at Kit25 NM 133730 4 . 59 1455033 _ at B430201 A12 Rik BB325849 4 .88 1443888 at AU023762 BB426608 + 1430519 _ a _ at Cnot7 AKO07767 4 .88 1438327 _ at Zfp533 BE981788 ? 1433451 _ at Cdk5r1 BB177836 4 .88 1433937 _ at Trp53bp2 BB814564 ? ? 1423202 _ a _ at Ncor1 U22016 4 .88 15 1454958 _ at Gsk3b BQ175580 ? 1455754 _ at Lmo3 BQ043839 87 1420667 _ at Doc2b BM117900 + ? 1425400 _ a _ at Cited4 BC025116 4 .87 1448922 at Dusp19 NM 024438 + 1422737 _ at Ncoa3 NM _ 008679 4 .87 1453008 _ at 2300002D11 Rik AK009004 4 . 57 1442363 at 1110012J17Rik BB461471 4 . 86 1418375 _ at Mbd6 NM 033072 4 . 56 1428698 _ at Dpp8 BM939621 4 .86 1417443 at Acot11 NM _ 025590 4 .56 1450071 _ at Ash11 BG694892 4 . 86 1425328 _ at BC008163 AU067643 4 .56 1447711 _ _ x _ at 4933412E12Rik BB265147 4 . 85 20 1456601 _ x _ at Fxyd2 AV002675 4 . 54 1450975 _ at Cacng4 BB333636 4 .84 1454845 _ x _ at Mchr1 AW049955 4 . 54 1451898 _ a _ at Sema?c AF363972 4 .84 1425327 _ at BC008163 AU067643 4 . 54 1426644 _ at Tbc1d20 BC002196 4 .83 1455772 at Pgr BB428874 4 . 54 1428872 _ at 4121402D02 Rik AW495537 4. 82 1450649 _ at Gng10 NM _ 025277 4 . 54 1417393 _ a _ at C1qdc2 NM _ 026125 4. 82 1435966 _ x _ at Mrpl13 BE953095 4 . 54 1453314 _ x _ at 2610039C10Rik AKO12533 4 . 80 25 1457088 _ at Pldn BB780781 4 .54 1450700 _ at Cdc42ep3 BB012489 4 .80 1432541 _ at 4930408017 Rik AK015114 4 .53 1455178 _ at Rutbol BB496468 4 .80 1422642 at Cdc42ep3 BB012489 4. 53 1424611 _ x _ at Trub2 BG064045 4 .79 1416606 _ S _ at Nola2 BC024944 4 .53 1435083 _ at Ctxn1 B1155559 4 .78 1423438 at Kptn AK009746 4 .53 1439422 _ a _ at C1qdc2 AV048291 4 . 78 1420871 _ at Gucylb3 BF472806 4 .52 1437707 _ at Slmol BB835597 4 .77 30 1417372 _ a at Peli1 BC016515 4 .52 1452533 at Ryr3 X83934 4 .77 1455170 _ at 2810001G20Rik BB250227 4 .51 1455114 _ at Conu BB547482 4 .77 1438889 _ at BB183456 4 . 51 1439031 at Jph4 BB376821 4 .76 1460058 _ at BE648595 4. 51 1451696 _ at Zfp64 BC004695 4 .76 1427674 _ a at Sezó D29763 4 .51 1424201 _ a _ at Seh11 AW537349 4 .76 1440204 _ at Foxgl AW494150 4 . 50 1436733 _ at E130309F12Rik BB523550 4 .75 35 1423699 _ at Ncaph2 BC003900 4 .50 1449241 _ at Klhli NM _ 053105 4 .75 1443372 _ at BB417900 4 .50 1435435 _ at Cttnbp2 BB357580 4 .74 1456858 _ at Gpr149 BB075339 4 . 49 1451867 _ x _ at Arhgapo AF177664 4 .73 1445605 _ S _ at 4921533L14Rik AI835491 4 .49 1453551 _ at Pola AKO20790 4 .73 1441048 at 5930430L01 Rik BB037822 4 . 49 1436563 _ at 4932441J04Rik AV277283 4 .73 1460343 _ at Neurl AF401228 4 . 49 1417530 a at Srp9 B1661964 4 .72 1421364 at Lrfn1 NM _ 030562 4 .49 1428357 _ at 2610019F03Rik AK011462 4 .72 40 1435666 _ at Mast3 AW553439 4 . 48 1424117 _ at BC056474 BCO24937 4 .72 1434295 _ at Rasgrp1 BE691356 4 . 48 1452063 _ at 2410081M15Rik AK010736 4 .72 1442562 _ at BB346556 4 .48 1430729 _ at Mygl AKO17955 4 .71 1418016 _ at Pum2 BI689507 4 .48 1435763 _ at Tbc1d16 BI218684 4 .71 1426585 _ s _ at Mapk1 BM209765 4 .47 1417893 _ at Sfxn3 NM _ 053197 4 . 70 1426450 _ at Plcl2 BM207017 4 .47 1460610 _ at 9430057019 Rik BG143491 4 . 70 45 1425558 _ at Klc3 BC017147 4 .47 1420094 _ at Hnrpdl AU015266 4 .69 1419838 _ s _ at Plk4 A1385771 4 . 46 1426676 _ s _ at Tomm70a BB225670 4 .69 1417178 _ at Gipc2 NM _ 016867 4 .46 1431520 _ at 4933406J09Rik AKO16696 4 .69 1454069 _ at 9030409C19Rik AKO18495 4 . 46 1425080 _ at Zfp286 BE651907 4 .68 1438955 _ x _ at Ppif AV209130 4 . 46 1428279 _ a _ at Atxn714 AKO13145 4 .68 1458399 _ at Lrrc3 A1413999 4 . 45 1453781 _ _ at AK009527 4 .68 50 1448553 _ at Myh7 NM _ 080728 4 . 45 1436013 _ at Gsg11 A1852434 4 .67 1453782 _ at 3021401C12Rik BI737125 4 .45 1441879 _ x _ at Mkrn1 AV218897 4 .67 1439552 _ at Trio BE985618 4 . 44 1454572 _ at 2810414N06Rik AK013093 4 .67 1450748 _ at Smpd3 NM _ 021491 4 .44 1435585 _ at Tceal7 BB378019 4 .67 1422799 _ at Bat2 AKO19427 4 . 44 1441051 _ at Trim27 BB401251 4 .67 1436827 _ at Gm944 BB053232 4 .44 1420580 _ at 4930429B21Rik NM _ 026249 4 .66 55 1440359 _ at 1700012H17Rik BB427277 4 . 44 1420446 _ at Odf3 NM _ 027019 4 .66 1427138 at 0610010D24Rik AW556861 4 .44 1450306 _ at Zp1 NM _ 009580 4 .66 1454772 _ at Ascc311 AI327392 4 .43 1437069 at Osbpl8 BG969333 4 .66 1435624 _ at 9030409G11Rik AV345336 4 . 42 1423835 _ at Zfp503 BB447914 4 . 66 1423186 _ at Tiam2 BM228957 4 . 42 1416397 _ at Mesdc1 BB474887 4 .65 1455548 _ at Dlgap4 BB314358 4 . 41 1425154 _ a _ at Csfi M21149 4 .65 1423973 _ a _ at Arf3 BC024935 4 . 41 1426893 _ at C230093N12 Rik BC023470 4 . 64 60 1421979 at Phex U73910 4 . 40 1426032 _ at Nfatc2 AF289078 4 . 63 1452609 at Cox4nb BI133691 4 . 40 1453577 _ at 2610018103Rik BB399635 4 .63 1419757 _ at Pitpnm2 NM _ 011256 4 . 40 1439888 _ at BB271581 4 .63 1455024 _ at Tlk1 AV269836 4 . 40 1436421 _ s _ at Arpc51 BB403233 4 .63 1439548 _ at Rap2b BB390705 4 . 40 1432312 _ a _ at 4931440B09Rik AK016509 4 .62 1460244 _ at Upb1 NM 133995 4 .40 1423831 _ at Prkag2 BB756794 4 .62 65 1435319 _ at Ihpk2 BB777667 4 . 39 1439246 _ x _ at Tnrcba BB822587 4 . 62 1451630 _ at Ttl BC018513 4 . 39 US 9, 816 ,096 B2 83 84 TABLE 13 -continued TABLE 13 - continued Genes differentially translated between medium Genes differentially translated between medium spiny neurons (MSNs ) and the rest of the brain . spiny neurons (MSNs ) and the rest of the brain . Probe Symbol GenBank FCH 5 Probe Symbol GenBank FCH 1440901 at Dgkb BB279524 4 . 38 1452984 _ at Ccny AK014507 4 . 18 1423563 _ at Prit1 AA739053 4 . 38 1451020 _ at Gsk3b BB831420 4 . 18 1426097 _ a _ at Ccdc106 BC018462 4 . 38 1448877 _ at Dlx2 NM 010054 4 . 17 1456755 _ at Trak1 A1467545 4 . 38 1455365 _ at Cdh8 BB426483 4 . 17 1423641 _ s _ at Cnot7 BC006021 4 . 38 10 1448498 at Rps6ka4 NM _ 019924 4 . 17 1424439 _ at 1810065E05Rik BC026941 4 . 38 1427261 _ at Wwcl BQ176786 4 .17 1429557 at Mcm8 AK010365 4 . 37 1423330 _ at Ensa AK006149 4 . 17 1415999 _ at Hey1 NM _ 010423 4. 37 1453278 _ a _ at Clip4 AV273445 4 . 16 1439632 _ at BQ174397 4 . 37 1432103 _ a _ at Sh3g13 AKO12114 4 . 16 1457149 _ at Ttc22 BB005679 4 . 36 1455102 _ at D330037H05Rik BB213860 4 .16 1423836 at Zfp503 BB447914 4 . 36 15 1451917 _ a at Dcamkli AF155820 4 .16 1448378 _ at Fscn1 NM _ 007984 4 . 35 1433657 _ at A130092J06Rik BI111848 4 .15 1421897 _ at Elk1 AI385733 4 .35 1442927 _ at Ptk2b BB548690 4 .15 1437001 at Gsk3b BQ173949 4 . 35 1430037 _ at Snx27 AKO17836 4 . 15 1435441 _ at Ablim2 BB336053 4 . 34 1449552 _ at Zfr NM 011767 4 . 15 1449110 _ at Rhob BC018275 4 .34 1435367 _ at Mapk4 BO177154 4 . 14 1429122 _ a _ at 1700040103Rik AK006658 4 . 34 1458711 _ at BB762344 4 . 14 1450623 _ at Gnb2 NM _ 010312 4 .33 20 1428214 _ at Tomm7 BB609428 4 . 14 1427607 _ at Cacnalh AB041801 4 .33 1427673 _ a at Semaze Z93948 4 . 14 1419924 at Fnip1 AW557298 4 .33 1427450 _ x _ at Myolb BIO80370 4 . 14 1426978 at Klh12 AW682368 4 .32 1456290 _ _ x _ at Ccm2 BB813044 4 . 13 1421193 _ a _ at Pbx3 NM _ 016768 4 .32 1432574 _ at 4930548F15 Rik BF466430 4 . 13 1438722 _ at 2610014116Rik A1854368 4 . 31 1423897 _ at Rnfl87 AW488376 4 . 13 1420834 _ at Vamp2 BG871810 4 . 31 25 1450570 _ aat Cd19 NM _ 009844 4 . 13 1425680 _ a __ at Btrc AF110396 4 . 31 1422977 _ at Gplbb NM _ 010327 4 . 12 1427930 _ at Pdxk BG063905 4 .31 1454844 _ at Mchr1 AW049955 4 . 12 1418161 _ _ at Jph3 NM _ 020605 4 . 30 1423785 at Eglnl BE995700 4 . 11 1450863 _ _ a __ at Dcamkl1 B0174703 4 . 30 1423012 _ at Syt7 NM _ 018801 4 . 11 1439957 _ at Gnal BF457720 4 . 29 1457116 _ at Rexol BI555624 4 . 10 1447530 _ at F8a BB274927 4 .29 30 1416391 at Ptcd1 NM _ 133735 4 . 10 1421260 _ a _ at Srm NM _ 009272 4 . 29 1456471 _ x _ at Phgdh BB204486 4 . 10 1441783 _ at Ccni BB749856 4 .28 1450142 _ a _ at Card14 NM _ 130886 4 .09 1428812 at 1700040L02Rik AK006661 4 .28 1442113 at 5330417C22Rik BB183350 4 .09 1452019 _ at Cyyrl AF442733 4 .28 1439986 _ at Dgki BE647270 4 . 09 1454939 _ at Phf2011 BB268102 4 . 28 1448709 at LOC669477 NM _ 033566 4 . 09 1420427 _ a _ at Dhx32 NM _ 133941 4 .27 25. 1423640 _ at Synpr BC026512 4 . 08 1455632 _ at Gnb5 BE446953 4 . 27 1458443 _ at Crtc1 BE986758 4 . 08 1448718 _ at 2400001E08Rik NM 025605 4 . 26 1441894 _ s _ at Grasp BB071890 4 .08 1432813 _ at 2900064F13Rik AK013749 4 . 26 1426065 _ a _ at Trib3 BC012955 4 . 08 1430135 _ at Dnase2a AKO18651 4 . 26 1417326 _ a _ at Anapc11 NM 025389 4 .08 1435845 _ at BB764479 4 . 26 1434569 _ at AA474455 BB428484 4 .08 1416508 _ at Med28 AK005130 4 . 26 1460392 _ a at Eny 2 BB610210 4 .07 1421199 _ at Dlg2 BB622767 4 . 26 40 1426354 _ at Bap1 AK009033 4 .07 1420887 _ a _ at Bcl211 NM _ 009743 4 .25 1457704 _ at Zfp533 BE982894 4 . 07 1439133 at 4930524007Rik AV267216 4 . 25 1458660 _ at A1414330 A1414330 4 .06 1417216 at Pim2 NM 138606 4 .25 1454721 _ at 1110018G07Rik AV257687 4 .06 1424037 _ at Itpka BC027291 4 .25 1434403 _ at Spred2 AV229054 4 . 06 1422683 _ at Iraklbp1 NM __ 022986 4 . 24 1455271 at LOC620695 BB560177 4 .05 1454902 _ at Prkcz BB430502 4 . 24 45 1420853 _ at Sdc3 BB528350 4 .05 1417233 _ at Chchd4 NM 133928 4 . 24 1431394 _ a _ at Lrrk2 AKO14938 4 .05 1418181 _ at pPtp4a3 AKO14601 4 . 24 1459983 _ at Impa2 AI115397 4 .05 1424384 _ a at Znrfi BC006765 4 . 24 1455620 _ at 4930551E15Rik BO177219 4 .04 1437306 _ at C130092011Rik BG071013 4 . 24 1454943 _ a _ at Paxipl BM935413 4 . 04 1417890 _ at Pdxp NM 020271 4 . 24 1434964 at X83328 BM952039 4 .04 1434943 _ at BC023055 BF464669 50 1438662 _ at Ajap1 AW123234 4 . 03 1430358 _ at Becn1 AV338072 4 . 23 1452974 _ at No18 AK017551 4 .03 1438211 _ s _ at Dbp BB550183 4 .23 1435169 _ at A930001N09Rik BB431047 4 .03 1437780 _ at Fancb AV222467 4 .23 1425271 at Psmc3ip AB000121 4 .03 1455865 _ at Insm1 BB468410 4 .22 1437642 _ at Hrbl BB243375 4 . 03 1456377 _ x _ at LOC632329 AV010467 4 .22 1419137 _ at Shank3 NM 021423 4 .03 1418893 at Pbx2 NM _ 017463 4 . 22 55 1423673 _ at Ldoc11 BC023053 4 . 03 1453324 _ at Nip7 BF459399 4 . 21 1448360 _ s _ at Angel2 NM 021421 4 . 03 1449063 _ at Sec22b BC009024 4 .21 1424523 at Elmo1 BC024727 4 . 02 1428951 _ at Nol8 AK017551 4 . 21 1455206 _ at C130006E23 BO175276 4 . 02 1422627 _ a _ at Mkks BF581250 4 . 21 1422527 _ at H2- DMa NM 010386 4 .02 1433816 _ at Mcart1 BQ031264 4 .21 1450928 at Id4 BB121406 4 .02 1439508 _ at Rab11b B1663913 4 . 21 1458351 _ s _ at Klh12 BB428573 4 . 01 1433955 at Brwd1 BB467195 4 .20 60 1422902 _ s _ at Mgea5 NM _ 023799 4 .01 1436128 _ at Plekha8 A1324154 4 . 20 1435942 _ at Kenq2 BE993301 4 .01 1458457 _ at BB750877 4 .20 1454960 _ at Smad3 BI646741 4 .00 1448150 _ at Nup62 NM _ 053074 4 . 20 1433854 _ at Tmem164 BG064061 4 .00 1460395 _ at Nudcd3 BB139222 4 . 19 1445653 _ at BC031361 BB825492 4 . 00 1434830 _ at Mxd1 AV228517 4 . 19 1442695 _ at C030007101Rik AW494399 4 .00 1451884 _ a _ at Lsm2 AF204156 4 . 19 65 1417192 _ at Tomm70a NM 138599 4 . 00 1447696 _ x _ at Adcy5 BB091427 4 . 18 1447301 _ at Akap5 BG228875 4 . 00 US 9, 816 ,096 B2 85 86 TABLE 13 -continued TABLE 13 - continued Genes differentially translated between medium Genes differentially translated between medium spiny neurons (MSNs ) and the rest of the brain . spiny neurons (MSNs ) and the rest of the brain . Probe Symbol GenBank FCH 5 Probe Symbol GenBank FCH 1426416 _ a _ at Yipf4 AV216410 4 . 00 1419352 _ at 17Rn6 BC003916 3 . 84 1438381 _ x _ at Ddx47 BB305306 4 . 00 1428443a at Raplgap AK005063 3 . 84 1425354 _ a _ at Aggf1 BC027286 4 . 00 1428723 _ at 2310047M1ORik AK009886 3 . 84 1420192 _ at D16Bwg1494e N28171 4 .00 1426887 _ at Nudt11 BQ174833 3 . 84 1458074 _ at EG328280 BB179240 3 . 99 10 1422439 _ a _ at Cdk4 NM _ 009870 3 . 84 1421355 _ at Tgm3 NM _ 009374 3 . 99 1442039 _ at Tox BF020502 3 . 84 1426031 a at Nfatc2 AF289078 3 .99 1419669 _ at Prtn3 U97073 3 . 83 1446498 _ at 1120ra BB551879 3 .99 1456970 _ at Cdh7 BF472513 3. 83 1434920 a at Evl AW553781 3 .99 1452295 _ at Tmepai AV291712 3 .82 1452531 _ _ at Runx1 X97306 3 . 99 1448511 _ _ at Ptprcap NM 016933 ??????3. 82 1452541 _ at Epb4 . 112 AJ245854 3 .98 15 . 1431646 _ a _ at Stx6 AK009690 3 .82 1417375 _ at Tuba4a AW491660 3 . 98 1448414 _ at Rad1 NM 011232 3 . 82 1439932 _ at 3110003A22Rik BQ174624 3 .98 1417355 _ at Peg3 ABO03040 3 . 82 1459299 _ at Myo3b AV381193 3 .98 1456441 _ at Rbm4b BB667164 3 .81 1420590 _ at Has1 NM 008215 3 .98 1417214 at Rab27b BB121269 3 . 81 1448990 _ a _ at Myolb A1255256 3 .98 1448554 _ s _ at Myh7 NM _ 080728 3 .81 1454092 _ a _ at Gtf2h3 AK017176 ??3 . 97 1453266 _ at Zbtb4 BB767069 3 . 81 1435638 _ at 9130221H12Rik BG808297 3 .97 20 1419611 _ _ at 4632415L05 Rik BC023403 3 . 81 1426580 _ at Plk4 BB706079 ????3. 97 1448919 _ at Cd302 NM 025422 3 . 80 1423174 _ a _ at Pard6b BE953582 3. 97 1429670 _ a _ at Lrriq2 BB321286 3 .80 1450655 _ at Pten AA214868 3 .97 1421975 _ a _ at Add2 AF189769 3 .79 1460214 _ at Pcp4 NM _ 008791 3 .97 1459054 _ at BC035954 BB496939 3 .79 1452281 _ at Sos2 Z11664 3. 97 1458339 _ at Cdadc1 BB231078 3 . 79 1429393 at Wdr40a B1558553 3 . 97 25 1427038 _ at Penk1 M13227 3 . 79 1422897 _ at Slc22a12 NM _ 009203 3 .96 1417844 _ at Med4 NM _ 026119 3 .79 1416125 at Fkbp5 U16959 3 .95 1434306 _ at Rab3ip BF319015 3 .78 1440213 _ a _ at 2010001M06Rik BE200030 3 . 95 1439764 _ s _ at Igf2bp2 BB098431 3 . 78 1420767 _ at 1700019G17Rik NM _ 029331 3 . 95 1435431 _ at 2310047M15 Rik AW558989 3 . 78 1423175 _ s _ at Pard6b BE953582 3 . 95 1420397 _ a _ at Spen NM 019763 3 .78 1435674 _ at Rhobtb2 BB351953 3 . 94 30 1433558 _ at Dab2ip BM231046 3 . 78 1434736 _ at Hlf BB744589 3 . 94 1420052 _ x _ at Psmb1 C81484 3 .78 1460205 _ at Dcakd NM _ 026551 3 .94 1439882 _ at Sec23ip BE685845 3 . 78 1422255 _ at Kcna4 NM _ 021275 3. 94 1443952 _ at Thra BI525006 3 .77 1433777 _ at L3mbt12 BB152370 3 . 94 1432378 _ at C030004G16Rik AK021043 3 .77 1439659 _ at Usp 49 BB014981 3 .94 1457897 _ at Iqce AV245518 ???????3 . 77 1427161 _ _ at Cenpf BE848253 3 . 94 35 1460671 _ at Gpx1 BI219063 3 .77 1448141 _ at 1110014J01Rik NM __ 029101 3 . 94 1431771 _ a _ at Iraklbp1 AKO14712 3 . 77 1418657 at Znhit4 BC019383 3 .93 1422609 at Arpp19 BE648432 3 . 77 1425006 _ a _ at Vrki BC016676 3 . 93 1427522 _ at Arhgap 20 BB623455 3 .76 1436408 _ at Rprml BE946298 3 .93 1426452 _ a _ at Rab30 BG070713 3 . 76 1453113 _ at Wdsub1 BB820363 3 .93 1452932 _ at Zbtb7a AK010379 3 . 76 1415904 _ at Lpl BC003305 3 . 92 1441495 _ at Kctd8 BB183090 3 .75 1426266 _ s _ at Zbtblos BB417508 3 .92 40 1419225 _ at Cacna2d3 NM _ 009785 3 . 75 1433860 _ at 6030458C11Rik BB126127 3 . 92 1434141 _ at Gucyla3 BG072799 3 .75 1427409 at 8 -Mar BB366341 3 . 92 1450662 _ at Teski NM 011571 3 .75 1436280 _ at Zfp31 BB757838 3 .92 1423217 _ aat 2510049119Rik AV109006 3 .75 1450853 _ at Tle4 AU045006 3 .91 1434277 _ a _ at Ypel2 BG069663 3 .75 1460436 _ at Ndst1 B1652065 3 .91 1430630 _ at BB236218 3 . 74 1424434 _ at BC024814 BCO24814 3 .91 45 1436986 _ at Sntb2 BB219478 3 . 74 1435043 _ at Plcb1 BB794831 3 . 90 1437006 X at Becnl C86082 3 .74 1458413 _ at Fbxw8 BB357976 3 . 90 1416603 _ at Rp122 NM _ 009079 3 .74 1454675 _ at Thra BI076689 3 . 90 1435525 _ at Kctd17 B1408602 3 . 74 1434795 _ at Disp1 BB174877 3 . 90 1437131_ _ x _ at Mrpl11 AV045020 3 . 74 1445854 _ at C230004F18 Rik BB380166 3 . 90 1453428 _ at 2700045P11 Rik AK012380 3 . 74 1420985 _ at Ash11 BG694892 3 . 90 50 1444808 _ at Pawr BG070555 3 .73 1426383 _ at Cry2 BF303057 3 . 90 1453083 _ at 6430701C03Rik AKO18294 3 .73 1448467 _ a _ at Ehbpili NM 053252 3 . 90 1435430 _ at Tmem1 BM200437 3 . 73 1437520 _ a _ at Nup85 BB320388 3 . 89 1457310 _ x _ at Scrt2 BG807042 3 .73 1421477 _ at Cplx2 NM _ 009946 3 . 89 1434082 _ at Pctk2 BM243464 3 .73 1417331 _ a _ at Arl6 NM _ _ 019665 3 . 88 1425920 _ at Cuedc1 BC025434 3. 73 1460738 _ at Limd2 AK012581 3 .88 55 1456106 _ x _ at Sdccag3 BB343189 3 .73 1417456 _ at Gnpat NM _ 010322 3 .88 1434805 _ at Mllt1 BG063049 3. 73 1454789 _ x _ at Prpf6 BB085604 3 .88 1422457 _ s _ at Sumo3 NM 019929 3 .72 1452628 _ at Bag5 AKO05534 3 . 87 1416113 _ at Fkbp8 NM 010223 3 . 72 1417075 _ at 2010309E21Rik NM 025591 3 . 87 1417462 _ at Cap1 NM 007598 3 . 72 1443225 _ at Acvrlc BB396526 3 . 87 1435825 _ at Acvrli BG969012 3 . 72 1438309 _ at BB432539 3 . 87 1437703 _ at EG382156 BM229128 3 . 71 1416744 _ at Uap1 NM _ 133806 3 .87 60 1428222 _ at Dcamkl2 AKO18179 3 . 71 1419166 _ at Slc5a2 BC022226 3 . 87 1451746 _ a _ at Atg12 AKO16474 3 . 71 1436795 at 9630058J23Rik BM247060 3 .86 1419224 _ at Cecr6 NM _ 033567 3 . 70 1448364 _ at Cong2 U95826 3 . 86 1457341 at BE991676 3 . 70 1427358 _ a _ at Dapk1 BCO26671 3 . 85 1428914 _ at 2310014D11 Rik AK009333 3 .70 1440505 at Plcxdi BB187908 3 .85 1418081_ at Wbscr18 NM 025362 3 .70 1433847 _ at D330017120Rik BB098407 ?? 65 1426521 _ at D230025D16Rik BG065702 3 . 70 1417114 _ at Gmcl1 AF163665 3 .85 1436887 _ x _ at Grwd1 BB251524 3 . 70