DOCSLIB.ORG

(12) Patent Application Publication (10) Pub. No.: US 2014/0196176 A1 Heintz Et Al

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
(12) Patent Application Publication (10) Pub. No.: US 2014/0196176 A1 Heintz Et Al US 2014O1961.76A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0196176 A1 Heintz et al. (43) Pub. Date: Jul. 10, 2014 (54) METHOD FOR ISOLATING CELL-TYPE (52) U.S. Cl. SPECIFICMRNAS CPC ........ CI2N 15/1041 (2013.01); C12N 15/1006 (2013.01); C12O 1/6827 (2013.01): CI2N (71) Applicants:Nathaniel Heintz, Pelham Manor, NY 15/82 (2013.01); C12N 15/8222 (2013.01) (US); Tito A. Serafini, San Mateo, CA USPC .......... 800/287: 530/358; 435/419,536/25.4; (US); Andrew W. Shyjan, San Carlos, 435/6.11:506/9; 435/6. 12:435/320.1; 800/298 CA (US) (72) Inventors: Nathaniel Heintz, Pelham Manor, NY (57) ABSTRACT (US); Tito A. Serafini, San Mateo, CA (US); Andrew W. Shyjan, San Carlos, The 1nVent1On provides methods for isolating cell-type spe CA (US) cific mRNAs by selectively isolating ribosomes or proteins that bind mRNA in a cell type specific manner, and, thereby, (21) Appl. No.: 13/930,864 the mRNA hound to the ribosomes or proteins that bind mRNA. Ribosomes, which are riboprotein complexes, bind (22) Filed: Jun. 28, 2013 mRNA that is being actively translated in cells. According to the methods of the invention, cells are engineered to express Related U.S. Application Data a molecularly tagged ribosomal protein or protein that binds (63) Continuation of application No. 13/104.316, filed on mRNA by introducing into the cell a nucleic acid comprising May 10, 2011, now Pat. No. 8,513,485, which is a a nucleotide sequence encoding a ribosomal protein or pro continuation of application No. 10/494.248, filed on tein that binds mRNA fused to a nucleotide sequence encod Aug. 16, 2004, now Pat. No. 7,985,553. ing a peptide tag. The tagged ribosome or mRNA binding protein can then be isolated, along with the mRNA bound to Publication Classification the tagged ribosome or mRNA binding protein, and the mRNA isolated and further used for gene expression analysis. (51) Int. Cl. The methods of the invention facilitate the analysis and quan CI2N IS/10 (2006.01) tification of gene expression in the selected cell type present CI2N 15/82 (2006.01) within a heterogeneous cell mixture, without the need to CI2O I/68 (2006.01) isolate the cells of that cell type as a preliminary step. Patent Application Publication Jul. 10, 2014 Sheet 1 of 2 US 2014/O1961.76 A1 Figure 1 1 2 3 4 5 Patent Application Publication Jul. 10, 2014 Sheet 2 of 2 US 2014/O1961.76 A1 Figure 2 ?iaeae US 2014/O 1961.76 A1 Jul. 10, 2014 METHOD FOR ISOLATING CELL-TYPE or other preparation containing the tagged protein that binds SPECIFICMRNAS mRNA being analyzed). In a preferred embodiment, the poly Some preparation is a membrane-associated polysome prepa CROSS-REFERENCE ration. Specifically, the peptide tag may be an epitope that is recognized by an antibody that does not specifically bind any 0001. This application is a continuation of application Ser. epitope expressed in a cell or ribosome/polysome fraction No. 13/104,316, filed May 10, 2011, which is a continuation from an unengineered cell. As defined herein, specific bind of application Ser. No. 10/494.248, filed on Aug. 16, 2004, ing is not competed away by addition of non-specific pro now U.S. Pat. No. 7,985,553, which is a national stage of teins, e.g., bovine serum albumen (BSA). The tagged riboso Application No. PCT/US02/34645, filed on Oct. 29, 2002, mal protein or mRNA binding protein is then expressed which claims priority to provisional Application No. 60/340, selectively in a cell population of interest (for example, by 689, filed on Oct. 29, 2001, each of which is incorporated by operably linking the nucleotide sequence encoding the tagged reference in its entirety. ribosomal protein or mRNA binding protein to a cell-type 1. TECHNICAL FIELD specific promoter and/or other transcriptional element). In a preferred embodiment, the tagged ribosomal protein or 0002 The present invention relates to methods for isolat mRNA binding protein is overexpressed. ing cell-type specific mRNAS by isolating ribosomes in a 0005 Monosomes or polysomes (which are, respectively, cell-type specific manner. According to the methods of the single or multiple ribosomes in a complex with a single invention, ribosomes or proteins that bind mRNA of the mRNA) or other mRNA-containing complex are isolated selected cell type are molecularly tagged and isolated, and the selectively from the cell population of interest through the use mRNA bound to the ribosomes or proteins that bind mRNA is of the tagged ribosomal protein subunit or other mRNA bind then isolated and analyzed. The methods of the invention ing protein. As used herein, isolated means that the ribosomes facilitate the analysis and quantification of gene expression in are separated from other cell components, specifically that the the selected cell type present within a heterogeneous cell ribosomes are substantially free of untagged ribosomes and mixture, without the need to isolate the cells of that cell type of RNA (particularly mRNA) not bound by ribosomes or as a preliminary step. mRNA binding protein. In particular, the composition is 50%, 2. BACKGROUND OF THE INVENTION 60%, 70%, 80%, 90%, 95% or 99% tagged ribosome or mRNA binding protein and associated mRNA. The mRNA 0003. An important paradigm in the development of new species that are bound to the cell-type specific ribosomes or diagnostics and therapies for human diseases and disorders is mRNA binding protein are then isolated, and can Subse the characterization of the gene expression of defined cell quently be profiled and quantified, to analyze gene expression types. The cellular complexity of many tissues (such as the in the cell. In a specific embodiment, because nascent nervous system), however, poses a challenge for those seek polypeptides are attached to isolated monosomes and poly ing to characterize gene expression at this level. The enor Somes, the methods of the invention can also be used to isolate mous heterogeneity of a tissue Such as the nervous system newly synthesized polypeptides from a cell type of interest (thousands of neuronal cell types, with non-neuronal cells (e.g., for proteomic applications), for example, using antibod outnumbering neuronal cells by an order of magnitude) is a ies that specifically recognize an epitope on a specific barrier to the identification and analysis of gene transcripts polypeptide being synthesized by the cell. present in individual cell types. One way to overcome this 0006. In preferred embodiments, the invention provides barrier is to tag gene transcripts directly or indirectly, i.e., transformed organisms (including animals, plants, fungi and mRNA, present in a particular cell type, in Such a manner as bacteria), e.g., a transgenic animal Such as a transgenic to allow facile isolation of the gene transcripts without the mouse, that expresses one or more tagged ribosomal protein need to isolate the individual cells of that cell type as a (s) or mRNA binding protein(s) within a chosen cell type. The preliminary step. We describe such a technology here. invention also provides cultured cells that express one or more tagged ribosomal proteins or mRNA binding proteins. 3. SUMMARY OF THE INVENTION Cell-type specific expression is achieved by driving the 0004. The invention provides methods for isolating cell expression of the tagged ribosomal protein using the endog type specific mRNAs by selectively isolating ribosomes or enous promoter of a particular gene, wherein the expression proteins that bind mRNA in a cell type specific manner, and, of the gene is a defining characteristic of the chosen cell type thereby, the mRNA bound to the ribosomes or proteins that (i.e., the promoter causes gene expression specifically in the bind mRNA. Ribosomes, which are riboprotein complexes, chosen cell type). Thus, “cell-type' refers to a population of bind mRNA that is being actively translated in cells. Accord cells characterized by the expression of a particular gene. In a ing to the methods of the invention, cells are engineered to preferred embodiment, a collection of transgenic mice express a molecularly tagged ribosomal protein or protein expressing tagged ribosomal proteins within a set of chosen that binds mRNA by introducing into the cell a nucleic acid cell types is assembled. Additionally, since the level of comprising a nucleotide sequence encoding a ribosomal pro expression of the tagged ribosomal protein or mRNA binding tein or proteins that bind mRNA fused to a nucleotide protein within a cell may be important in the efficiency of the sequence encoding a peptide tag. The peptide tag can be any isolation procedure, in certain embodiments of the invention, non-ribosomal protein peptide or non-mRNA binding protein a binary system can be used, in which the endogenous pro peptide that is specifically bound by a reagent that either does moter drives expression of a protein that then activates a not recognize a component of the cell fraction from which the second expression construct. This second expression con tagged ribosomes or proteins that bind mRNA are to be iso structuses a strong promoter to drive expression of the tagged lated, for example, from a whole cell lysate or post-mitochon ribosomal protein or mRNA binding protein at higher levels drial fraction (or any other ribosome or polysome preparation than is possible using the endogenous promoter itself. US 2014/O 1961.76 A1 Jul. 10, 2014 0007. In specific embodiments, the invention provides does not recognize a component, other than the peptide tag, of molecularly tagged ribosomes, preferably bound to mRNA, the cell fraction from which the tagged ribosomes or mRNA that are bound to an affinity reagent for the molecular tag.
Load more

Recommended publications
  • Supplementary Materials
    Supplementary materials Supplementary Table S1: MGNC compound library Ingredien Molecule Caco- Mol ID MW AlogP OB (%) BBB DL FASA- HL t Name Name 2 shengdi MOL012254 campesterol 400.8 7.63 37.58 1.34 0.98 0.7 0.21 20.2 shengdi MOL000519 coniferin 314.4 3.16 31.11 0.42 -0.2 0.3 0.27 74.6 beta- shengdi MOL000359 414.8 8.08 36.91 1.32 0.99 0.8 0.23 20.2 sitosterol pachymic shengdi MOL000289 528.9 6.54 33.63 0.1 -0.6 0.8 0 9.27 acid Poricoic acid shengdi MOL000291 484.7 5.64 30.52 -0.08 -0.9 0.8 0 8.67 B Chrysanthem shengdi MOL004492 585 8.24 38.72 0.51 -1 0.6 0.3 17.5 axanthin 20- shengdi MOL011455 Hexadecano 418.6 1.91 32.7 -0.24 -0.4 0.7 0.29 104 ylingenol huanglian MOL001454 berberine 336.4 3.45 36.86 1.24 0.57 0.8 0.19 6.57 huanglian MOL013352 Obacunone 454.6 2.68 43.29 0.01 -0.4 0.8 0.31 -13 huanglian MOL002894 berberrubine 322.4 3.2 35.74 1.07 0.17 0.7 0.24 6.46 huanglian MOL002897 epiberberine 336.4 3.45 43.09 1.17 0.4 0.8 0.19 6.1 huanglian MOL002903 (R)-Canadine 339.4 3.4 55.37 1.04 0.57 0.8 0.2 6.41 huanglian MOL002904 Berlambine 351.4 2.49 36.68 0.97 0.17 0.8 0.28 7.33 Corchorosid huanglian MOL002907 404.6 1.34 105 -0.91 -1.3 0.8 0.29 6.68 e A_qt Magnogrand huanglian MOL000622 266.4 1.18 63.71 0.02 -0.2 0.2 0.3 3.17 iolide huanglian MOL000762 Palmidin A 510.5 4.52 35.36 -0.38 -1.5 0.7 0.39 33.2 huanglian MOL000785 palmatine 352.4 3.65 64.6 1.33 0.37 0.7 0.13 2.25 huanglian MOL000098 quercetin 302.3 1.5 46.43 0.05 -0.8 0.3 0.38 14.4 huanglian MOL001458 coptisine 320.3 3.25 30.67 1.21 0.32 0.9 0.26 9.33 huanglian MOL002668 Worenine
    [Show full text]
  • Food Deprivation Explains Effects of Mouthbrooding on Ovaries and Steroid Hormones, but Not Brain Neuropeptide and Receptor Mrnas, in an African Cichlid fish
    Hormones and Behavior 62 (2012) 18–26 Contents lists available at SciVerse ScienceDirect Hormones and Behavior journal homepage: www.elsevier.com/locate/yhbeh Food deprivation explains effects of mouthbrooding on ovaries and steroid hormones, but not brain neuropeptide and receptor mRNAs, in an African cichlid fish Brian P. Grone ⁎,1, Russ E. Carpenter, Malinda Lee, Karen P. Maruska, Russell D. Fernald Biology Department, Stanford University, Stanford, CA 94305‐5020, USA article info abstract Article history: Feeding behavior and reproduction are coordinately regulated by the brain via neurotransmitters, circulating Received 19 December 2011 hormones, and neuropeptides. Reduced feeding allows animals to engage in other behaviors important for Revised 10 April 2012 fitness, including mating and parental care. Some fishes cease feeding for weeks at a time in order to provide Accepted 15 April 2012 care to their young by brooding them inside the male or female parent's mouth. Maternal mouthbrooding is Available online 26 April 2012 known to impact circulating hormones and subsequent reproductive cycles, but neither the full effects of food deprivation nor the neural mechanisms are known. Here we ask what effects mouthbrooding has on Keywords: Feeding several physiological processes including gonad and body mass, brain neuropeptide and receptor gene ex- Reproduction pression, and circulating steroid hormones in a mouthbrooding cichlid species, Astatotilapia burtoni.We Parental care ask whether any observed changes can be explained by food deprivation, and show that during mouthbrood- Teleost ing, ovary size and circulating levels of androgens and estrogens match those seen during food deprivation. Cichlid Levels of gonadotropin-releasing hormone 1 (GnRH1) mRNA in the brain were low in food-deprived females Testosterone compared to controls and in mouthbrooding females compared to gravid females.
    [Show full text]
  • Targeting EZH2 Increases Therapeutic Efficacy of PD-1 Check-Point Blockade in Models of Prostate Cancer Supplement Figures and T
    Targeting EZH2 Increases Therapeutic Efficacy of PD-1 Check-Point Blockade in Models of Prostate Cancer Supplement Figures and Tables 1 Fig. S1. (A) Schema and genotyping PCR example for the creation of EM and EMC genetically engineered mice. (B) Three-dimensional PCa organoids generated from EM mice (without PSACreERT2) alleles. When treated with tamoxifen, demonstrates no loss of H3K27me3 or EDU staining, indicating specificity of tamoxifen-PSACreERT2 mediated deletion of the Ezh2 set domain. (C) Principle component analysis (PCA) following chemical and genetic inhibition of Ezh2 catalytic function results in significant changes in gene expression. 2 Fig. S2. (A) A 29-gene signature derived from Fig. 1C demonstrates complete independence from a previously published polycomb repression signature. (B) Our 29 gene signature demonstrates significant correlation with a previously published polycomb repression signature in 2 independent human PCa gene expression datasets. (C) EZH2 activity is not determined by EZH2 mRNA expression. 3 Fig. S3. A 29-gene signature derived from Fig 1C was used to generate signature scores for each patient within four independent human prostate cancer RNA-seq datasets. Patients were ranked highest score to lowest score and subject to quartile separation. First (blue) and fourth (red) quartiles were analyzed by supervised clustering to demonstrate expression differences within patients with most lowest EZH2 activity and most highest EZH2 activity. 4 Fig. S4. Genes representing IFN signaling (STAT1, IRF9), Th1 chemokines (CXCL10, CXCL11), and MHC Class I molecules (B2M, HLA-A) were shown to be enriched in PCa patients with low EZH2 activity. 5 Fig. S5. Treatment of 22Rv1 human 2D cell lines with the demonstrated conditions for 96 hours show that EZH2 inhibition increases expression of dsRNA (green = dsRNA, blue = nuclei).
    [Show full text]
  • (12) Patent Application Publication (10) Pub. No.: US 2003/0106074 A1 Serafini (43) Pub
    US 2003O106074A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2003/0106074 A1 Serafini (43) Pub. Date: Jun. 5, 2003 (54) COLLECTIONS OF TRANSGENIC ANIMAL Publication Classification LINES (LIVING LIBRARY) (51) Int. Cl." ...................... A01K 67/033; AO1K 67/027 (76) Inventor: Tito Andrew Serafini, San Mateo, CA (52) U.S. Cl. ................................................... 800/8: 800/14 (US) (57) ABSTRACT Correspondence Address: The invention provides collections of transgenic animals and PENNIE AND EDMONDS vectors for producing transgenic animals, which transgenic 1155 AVENUE OF THE AMERICAS animals and vectors have a transgene comprising Sequences NEW YORK, NY 100362711 encoding a detectable or Selectable marker, the expression of which marker is under the control of regulatory Sequences (21) Appl. No.: 10/077,025 from an endogenous gene Such that when the transgene is present in the genome of the transgenic animal, the detect (22) Filed: Feb. 14, 2002 able or Selectable marker has the same expression pattern as the endogenous gene. Such transgenic animals can then be Related U.S. Application Data used to detect, isolate and/or Select pure populations of cells having a particular functional characteristic. The isolated (63) Continuation-in-part of application No. 09/783,487, cells have uses in gene discovery, target identification and filed on Feb. 14, 2001. validation, genomic and proteomic analysis, etc. Patent Application Publication Jun. 5, 2003. Sheet 1 of 13 US 2003/0106074 A1 sixxx; : ?,graecaeaeaeaeae ·:>`()~(ºrº?anaeru: !!¿*(:,!!!!!(!!!!!..”):straes) - k wis **** ************ FIG . 1 A FIG. 1B Patent Application Publication Jun. 5, 2003. Sheet 2 of 13 US 2003/0106074 A1 clone2 *********** ******$$x***** *******.
    [Show full text]
  • The DNA Sequence and Comparative Analysis of Human Chromosome 20
    articles The DNA sequence and comparative analysis of human chromosome 20 P. Deloukas, L. H. Matthews, J. Ashurst, J. Burton, J. G. R. Gilbert, M. Jones, G. Stavrides, J. P. Almeida, A. K. Babbage, C. L. Bagguley, J. Bailey, K. F. Barlow, K. N. Bates, L. M. Beard, D. M. Beare, O. P. Beasley, C. P. Bird, S. E. Blakey, A. M. Bridgeman, A. J. Brown, D. Buck, W. Burrill, A. P. Butler, C. Carder, N. P. Carter, J. C. Chapman, M. Clamp, G. Clark, L. N. Clark, S. Y. Clark, C. M. Clee, S. Clegg, V. E. Cobley, R. E. Collier, R. Connor, N. R. Corby, A. Coulson, G. J. Coville, R. Deadman, P. Dhami, M. Dunn, A. G. Ellington, J. A. Frankland, A. Fraser, L. French, P. Garner, D. V. Grafham, C. Grif®ths, M. N. D. Grif®ths, R. Gwilliam, R. E. Hall, S. Hammond, J. L. Harley, P. D. Heath, S. Ho, J. L. Holden, P. J. Howden, E. Huckle, A. R. Hunt, S. E. Hunt, K. Jekosch, C. M. Johnson, D. Johnson, M. P. Kay, A. M. Kimberley, A. King, A. Knights, G. K. Laird, S. Lawlor, M. H. Lehvaslaiho, M. Leversha, C. Lloyd, D. M. Lloyd, J. D. Lovell, V. L. Marsh, S. L. Martin, L. J. McConnachie, K. McLay, A. A. McMurray, S. Milne, D. Mistry, M. J. F. Moore, J. C. Mullikin, T. Nickerson, K. Oliver, A. Parker, R. Patel, T. A. V. Pearce, A. I. Peck, B. J. C. T. Phillimore, S. R. Prathalingam, R. W. Plumb, H. Ramsay, C. M.
    [Show full text]
  • Graded Co-Expression of Ion Channel, Neurofilament, and Synaptic Genes in Fast- Spiking Vestibular Nucleus Neurons
    Research Articles: Cellular/Molecular Graded co-expression of ion channel, neurofilament, and synaptic genes in fast- spiking vestibular nucleus neurons https://doi.org/10.1523/JNEUROSCI.1500-19.2019 Cite as: J. Neurosci 2019; 10.1523/JNEUROSCI.1500-19.2019 Received: 26 June 2019 Revised: 11 October 2019 Accepted: 25 October 2019 This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data. Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2019 the authors 1 Graded co-expression of ion channel, neurofilament, and synaptic genes in fast-spiking 2 vestibular nucleus neurons 3 4 Abbreviated title: A fast-spiking gene module 5 6 Takashi Kodama1, 2, 3, Aryn Gittis, 3, 4, 5, Minyoung Shin2, Keith Kelleher2, 3, Kristine Kolkman3, 4, 7 Lauren McElvain3, 4, Minh Lam1, and Sascha du Lac1, 2, 3 8 9 1 Johns Hopkins University School of Medicine, Baltimore MD, 21205 10 2 Howard Hughes Medical Institute, La Jolla, CA, 92037 11 3 Salk Institute for Biological Studies, La Jolla, CA, 92037 12 4 Neurosciences Graduate Program, University of California San Diego, La Jolla, CA, 92037 13 5 Carnegie Mellon University, Pittsburgh, PA, 15213 14 15 Corresponding Authors: 16 Takashi Kodama ([email protected]) 17 Sascha du Lac ([email protected]) 18 Department of Otolaryngology-Head and Neck Surgery 19 The Johns Hopkins University School of Medicine 20 Ross Research Building 420, 720 Rutland Avenue, Baltimore, Maryland, 21205 21 22 23 Conflict of Interest 24 The authors declare no competing financial interests.
    [Show full text]
  • Rabbit Anti-CLCA3 Antibody-SL13706R
    SunLong Biotech Co.,LTD Tel: 0086-571- 56623320 Fax:0086-571- 56623318 E-mail:[email protected] www.sunlongbiotech.com Rabbit Anti-CLCA3 antibody SL13706R Product Name: CLCA3 Chinese Name: 钙激活氯离子Channel protein3抗体 Calcium activated chloride channel 3; Calcium-activated chloride channel regulator family member 3; Chloride channel accessory 3 pseudogene; chloride channel calcium Alias: activated 3; Chloride channel calcium activated family member 3; Clca1; Clca2; CLCA3P; Gob 5; Gob-5; Gob5; hCLCA3; mCLCA3; MGC143984; MGC143985. Organism Species: Rabbit Clonality: Polyclonal React Species: Mouse,Rat,Dog, WB=1:500-2000ELISA=1:500-1000IHC-P=1:400-800IHC-F=1:400-800ICC=1:100- 500IF=1:100-500（Paraffin sections need antigen repair） Applications: not yet tested in other applications. optimal dilutions/concentrations should be determined by the end user. Molecular weight: 98kDa Cellular localization: cytoplasmic Form: Lyophilized or Liquid Concentration: 1mg/ml immunogen: KLHwww.sunlongbiotech.com conjugated synthetic peptide derived from mouse CLCA3:811-913/913 Lsotype: IgG Purification: affinity purified by Protein A Storage Buffer: 0.01M TBS(pH7.4) with 1% BSA, 0.03% Proclin300 and 50% Glycerol. Store at -20 °C for one year. Avoid repeated freeze/thaw cycles. The lyophilized antibody is stable at room temperature for at least one month and for greater than a year Storage: when kept at -20°C. When reconstituted in sterile pH 7.4 0.01M PBS or diluent of antibody the antibody is stable for at least two weeks at 2-4 °C. PubMed: PubMed The calcium-activated chloride channel (CLCA) protein family, which includes the human homologs CLCA1 and CLCA2, display distinct tissue distribution patterns.
    [Show full text]
  • Gnrhr2 (67-R): Sc-100301
    SANTA CRUZ BIOTECHNOLOGY, INC. GnRHR2 (67-R): sc-100301 BACKGROUND PRODUCT Gonadotropin-releasing hormone (GnRH) is released in a pulsatile manner that Each vial contains 100 µg IgG2a kappa light chain in 1.0 ml of PBS with varies with the reproductive cycle. This hypothalamic hormone is transported < 0.1% sodium azide and 0.1% gelatin. to the pituitary, where it binds to specific receptors and regulates the synthe- sis and release of luteinizing hormone (LH) and follicle-stimulating hormone APPLICATIONS (FSH). GnRHR2 (Gonadotropin-releasing hormone (type 2) receptor 2), also GnRHR2 (67-R) is recommended for detection of GnRHR2 of human origin known as GnRH II-R, is a 379 amino acid multi-pass membrane protein that by Western Blotting (starting dilution 1:200, dilution range 1:100-1:1000), is expressed in a variety of tissues, where it functions as a G protein-coupled immunoprecipitation [1-2 µg per 100-500 µg of total protein (1 ml of cell receptor for GnRH. Localized to the cell membrane, GnRHR2 mediates its own lysate)] and solid phase ELISA (starting dilution 1:30, dilution range 1:30- receptor activity via association with G proteins, thereby activating a phospha- 1:3000). tidylinositol-calcium second messenger system that regulates GnRHR2 func- tion. GnRHR2 is thought to have potent antiproliferative effects on ovarian and Suitable for use as control antibody for GnRHR2 siRNA (h): sc-108007, endometrial cancer cells, suggesting a possible role in tumor suppression. Due GnRHR2 shRNA Plasmid (h): sc-108007-SH and GnRHR2 shRNA (h) Lenti- to alternative splicing events, GnRHR2 is expressed as two isoforms.
    [Show full text]
  • Views of the NIDA, NINDS Or the National Summed Across the Three Auditory Forebrain Lobule Sec- Institutes of Health
    Xie et al. BMC Biology 2010, 8:28 http://www.biomedcentral.com/1741-7007/8/28 RESEARCH ARTICLE Open Access The zebra finch neuropeptidome: prediction, detection and expression Fang Xie1, Sarah E London2,6, Bruce R Southey1,3, Suresh P Annangudi1,6, Andinet Amare1, Sandra L Rodriguez-Zas2,3,5, David F Clayton2,4,5,6, Jonathan V Sweedler1,2,5,6* Abstract Background: Among songbirds, the zebra finch (Taeniopygia guttata) is an excellent model system for investigating the neural mechanisms underlying complex behaviours such as vocal communication, learning and social interactions. Neuropeptides and peptide hormones are cell-to-cell signalling molecules known to mediate similar behaviours in other animals. However, in the zebra finch, this information is limited. With the newly-released zebra finch genome as a foundation, we combined bioinformatics, mass-spectrometry (MS)-enabled peptidomics and molecular techniques to identify the complete suite of neuropeptide prohormones and final peptide products and their distributions. Results: Complementary bioinformatic resources were integrated to survey the zebra finch genome, identifying 70 putative prohormones. Ninety peptides derived from 24 predicted prohormones were characterized using several MS platforms; tandem MS confirmed a majority of the sequences. Most of the peptides described here were not known in the zebra finch or other avian species, although homologous prohormones exist in the chicken genome. Among the zebra finch peptides discovered were several unique vasoactive intestinal and adenylate cyclase activating polypeptide 1 peptides created by cleavage at sites previously unreported in mammalian prohormones. MS-based profiling of brain areas required for singing detected 13 peptides within one brain nucleus, HVC; in situ hybridization detected 13 of the 15 prohormone genes examined within at least one major song control nucleus.
    [Show full text]
  • Analysis of the Mouse Transcriptome for Genes Involved in the Function of the Nervous System Stefano Gustincich,1,13,14 Serge Batalov,2 Kirk W
    Downloaded from genome.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press Letter Analysis of the Mouse Transcriptome for Genes Involved in the Function of the Nervous System Stefano Gustincich,1,13,14 Serge Batalov,2 Kirk W. Beisel,3 Hidemasa Bono,4 Piero Carninci,4,5 Colin F. Fletcher,2,6 Sean Grimmond,7 Nobutaka Hirokawa,8 Erich D. Jarvis,9 Tim Jegla,2 Yuka Kawasawa,10 Julianna LeMieux,1 Harukata Miki,8 Elio Raviola,1 Rohan D. Teasdale,7 Naoko Tominaga,4 Ken Yagi,4 Andreas Zimmer,11 RIKEN GER Group4 and GSL Members,5,12 Yoshihide Hayashizaki,4,5 and Yasushi Okazaki4,5 1Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, USA; 2Genomics Institute of the Novartis Research Foundation (GNF), San Diego, California 92121, USA; 3Boys Town National Research Hospital, Omaha, Nebraska 68131, USA; 4Laboratory for Genome Exploration Research Group, RIKEN Genomic Sciences Center (GSC), RIKEN Yokohama Institute, Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan; 5Genome Science Laboratory, RIKEN, Hireosawa, Wako, Saitama, 351-0198, Japan; 6The Scripps Research Institute, La Jolla, California 92037, USA; 7Institute for Molecule Bioscience and ARC Special Research Centre for Functional and Applied Genomics, University of Queensland, Q4072, Australia; 8Graduate School of Medicine, University of Tokyo, Tokyo, 113-0033, Japan; 9Duke University Medical Center, Department of Neurobiology, Durham, North Carolina 27710, USA; 10Howard Hughes Medical Institute, Department of Molecular Genetics,
    [Show full text]
  • Kisspeptin and Gonadotropin-Releasing Hormone Signaling in Skeletal Muscle
    KISSPEPTIN AND GONADOTROPIN-RELEASING HORMONE SIGNALING IN SKELETAL MUSCLE A Thesis Presented to the Faculty of the Graduate School of Cornell University In Partial Fulfillment of the Requirements for the Degree of Master of Science by Lauren Mary Varvatos August 2019 © 2019 Lauren Mary Varvatos ABSTRACT Chronic inflammation contributes to skeletal muscle atrophy and impaired regenerative capacity. Previous research has identified skeletal muscle inflammatory susceptibility (MuIS), or the ability to manage and respond to inflammation, as a predictor of failed skeletal muscle regeneration and regrowth following surgery. This inflammatory susceptibility is associated with human aging and likely contributes to the adverse structural, metabolic, and functional tissue remodeling that occurs in aged adults. The etiology for heightened inflammation in skeletal muscle is unclear, particularly in healthy young adults. Research that elucidates the mechanisms by which inflammation impairs skeletal muscle regeneration is necessary for identifying potential therapeutic targets. Previous research from our laboratory identified kisspeptin-1 (KISS1) as a highly differentially expressed gene (DEgene) associated with lower inflammatory susceptibility (MuIS-) in human skeletal muscle. When the MuIS- group was compared to a group with an improved hypertrophic response to resistance training, gonadotropin-releasing hormone (GnRH) signaling emerged as a top canonical pathway. In the present study, we aimed to investigate the potential relationship between KISS1/GnRH signaling and inflammation in skeletal muscle and elucidate the relevant signaling pathways. The presence of the GnRH receptor (GnRHr) was assessed in the skeletal muscle of mice genetically modified to constitutively express β-galactosidase at the GnRHr promoter (RG) and wild type (WT) mice using a β-galactosidase gene detection assay.
    [Show full text]
  • (12) United States Patent (10) Patent No.: US 7,666,610 B2 Saitoh Et Al
    USOO766661 OB2 (12) United States Patent (10) Patent No.: US 7,666,610 B2 Saitoh et al. (45) Date of Patent: Feb. 23, 2010 (54) EXPRESSING TRANSPORTERS ON VIRAL JP 11-172 1, 1999 ENVELOPES JP 2001-197846 T 2001 JP 2001-1394.96 5, 2005 (75) Inventors: Ryoichi Saitoh, Shizuoka (JP); KR 99071666 9, 1999 Toshihiko Ohtomo, Shizuoka (JP); WO WO97, 19919 6, 1997 Masayuki Tsuchiya, Shizuoka (JP) WO WO 98.46777 10, 1998 WO WOOO.28O16 5, 2000 (73) Assignee: Chugai Seiyaku Kabushiki Kaisha, WO WOO3/033024 4/2003 Tokyo (JP) WO WO 03/0476.21 6, 2003 WO WO 03/08311.6 A1 10, 2003 (*) Notice: Subject to any disclaimer, the term of this WO WOO3/104453 12/2003 patent is extended or adjusted under 35 U.S.C. 154(b) by 283 days. OTHER PUBLICATIONS (21) Appl. No.: 10/509,343 Hsu et al., Overexpression of human intestinal oligopeptide trans porter in mammalian cells via adenoviral transduction, 1998, Phar (22) PCT Filed: Mar. 28, 2003 maceutical Research, vol. 15, No. 9, pp. 1376-1381.* Garcia et al., cDNA cloning of MCT2, a second monocarboxylate (86). PCT No.: PCT/UP03/03975 transporter expressed in different cells than MCT1, 1995, Journal of Biological Chemistry, vol. 270, No. 4, pp. 1843-1849.* S371 (c)(1), Miyasaka, et al., Characterization of human taurine transporter (2), (4) Date: Jun. 21, 2005 expressed in insect cells using recombinant baculovirus, 2001, Pro tein Expression and Purification, vol. 23, pp. 389-397.* (87) PCT Pub. No.: WO03/083116 Hsu et al., Overexpression of human intestinal oligopeptide trans porter in mammalian cells via adenoviral transduction, 1998, Phar PCT Pub.
    [Show full text]