|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 ) .