DOCSLIB.ORG

Gene Trapping in Differentiating Cell Lines: Regulation of the Lysosomal Protease Cathepsin B in Skeletal Myoblast Growth and Fusion Joseph A

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Gene Trapping in Differentiating Cell Lines: Regulation of the Lysosomal Protease Cathepsin B in Skeletal Myoblast Growth and Fusion Joseph A Gene Trapping in Differentiating Cell Lines: Regulation of the Lysosomal Protease Cathepsin B in Skeletal Myoblast Growth and Fusion Joseph A. Gogos,* Rachel Thompson,* William Lowry,* Bonnie F. Sloane, ~ Harold Weintraub,** and Marshall Horwitz ~ *Howard Hughes Medical Institute, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109;*Department of Pharmacology, Wayne State University, Detroit, Michigan 48201; and ~Markey Molecular Medicine Center, Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, Washington 98195 Abstract. To identify genes regulated during skeletal and some show specific patterns of subcellular localiza- muscle differentiation, we have infected mouse C2C12 tion. The first gene we have identified with this strategy myoblasts with retroviral gene trap vectors, containing is the lysosomal cysteine protease cathepsin B. Expres- a promoterless marker gene with a 5' splice acceptor sion from the trapped allele is upregulated during early signal. Integration of the vector adjacent to an actively myoblast fusion and downregulated in myotubes. A di- transcribed gene places the marker under the transcrip- rect role for cathepsin B in myoblast growth and fusion tional control of the endogenous gene, while the adja- is suggested by the observation that the trapped cells cent vector sequences facilitate cloning. The vector in- deficient in cathepsin B activity have an unusual mor- sertionally mutates the trapped locus and may also phology and reduced survival in low-serum media and form fusion proteins with the endogenous gene prod- undergo differentiation with impaired cellular fusion. uct. We have screened several hundred clones, each The phenotype is reproduced by antisense cathepsin B containing a trapping vector integrated into a different expression in parental C2C12 myoblasts. The cellular endogenous gene. In agreement with previous esti- phenotype is similar to that observed in cultured myo- mates based on hybridization kinetics, we find that a blasts from patients with I cell disease, in which there is large proportion of all genes expressed in myoblasts are diminished accumulation of lysosomal enzymes. This regulated during differentiation. Many of these genes suggests that a specific deficiency of cathepsin B could undergo unique temporal patterns of activation or re- contribute to the myopathic component of this illness. pression during cell growth and myotube formation, HE expression of a myogenic basic helix-100p-helix netics (Leibovitch et al., 1979). It was estimated that (bHLH) 1 transcription factor of the MyoD family is ~30,0OO genes are expressed in skeletal myoblasts, and T sufficient to convert a variety of cultured cells into about two-thirds of those are regulated during the course skeletal muscle (for review see Mtinsterberg and Lassar, of differentiation to myotubes. 1994), This initial switch is followed by an irreversible cas- Gene trap vectors provide an alternative way to study cade of gene activation and repression events underlying both global and gene-specific changes in transcription and the morphological differentiation. Earlier studies have an- mRNA accumulation (Wurst et al., 1995; Skarnes et al., alyzed global changes in sequence complexity and fre- 1995; DeGregori et al., 1994; Friedrich and Soriano, 1993). quency distribution of messenger RNAs during muscle The vector that we used contains a promoterless marker differentiation in vitro using DNA-RNA hybridization ki- gene with a 5' splice acceptor signal. Integration of the vector adjacent to an actively transcribed gene places the *Dr. Harold Weintraub died on 28 March 1995. marker under the control of the endogenous transcription Address all correspondence to J.A. Gogos at his present address: Cen- unit and facilitates its cloning. There are advantages com- ter for Neurobiology and Behaviour, College of Physicians and Surgeons, pared to other approaches used to study gene induction or Columbia University, 701 West 168th Street, New York, NY 10032. repression, such as subtractive hybridization or differential E-mail: [email protected]; or to M. Horowitz, Markey Mo- lecular Medicine Center, Division of Medical Genetics, Department of display. First, the trapping event is, in principle, indepen- Medicine, Box 357720, University of Washington, Seattle, WA 98195. dent of the abundance of the message, potentially allowing Tel.: (206) 616-4566. Fax: (206) 616-7288. E-mail: [email protected] the identification of mRNA that exist in low numbers. ton.edu Even differential display, the most sensitive of the hybrid- 1. Abbreviations used in this paper: bHLH, basic helix-100p-helix. ization methods, shows a strong bias towards high copy © The Rockefeller University Press, 0021-9525/96/08/837/11 $2.00 The Journal of Cell Biology, Volume 134, Number 4, August 1996 837-847 837 number transcripts (Bertioli et al., 1995). Second, the gene observed preferential amplification of the 5' product, the protocol was trap vectors may generate fusion products between the re- modified as follows: 3 l~g of genomic DNA were digested to completion with 20 U of HhaI in 1X PCR buffer (50 mM KCI, 10 mM Tris, pH 8.5, 2 porter gene and part of the endogenous gene, which could mM MgCI2, 0.01% gelatin) in the presence of 0.01 Ixg RNase A in a final include a subcellular localization signal, thereby providing volume of 100 Ixl. After heat inactivation of the enzyme, one third of the information on the localization of the host encoded pro- restriction digest was used for a subsequent self-ligation step, in a total tein. Third, although integration of the gene trap is most volume of 100 ixl adjusted with 1X PCR buffer, 1 Ixl of 10 mM ATP, and 1 Ixl likely to result in a recessive loss of function mutation, of T4 DNA ligase (5 Weiss U//.tl). Ligation was performed for 15 rain at 37°C followed by an overnight incubation at room temperature. Ligase some genes may be vulnerable to haploinsufficiency, and was inactivated for 10 rain at 68°C, and 50 Ixl were digested with 20 U of some established cell lines are hypodiploid (Siminovitch, XbaI for 60 min at 37°C. About one fifth of the restriction digest was used 1976). Integrations into such genes could result in a mu- for a first round of PCR using two external primers, A (TCCATGCCT- tant phenotype and provide additional information on the TGCAAAATGGC) and B (GCGGCGGCCGCATGACCCTGTGCCT- TATT). First round of amplification was done in a 50-pA total volume con- function of the gene. taining 5 Ixl of 10× PCR buffer, 1 mM dNTPs, a 1-1xM concentration of We have therefore sought a strategy to identify and each primer, and 2 U of AmpliTaq DNA polymerase (Perkin Elmer clone genes regulated during skeletal muscle differentia- Corp., Norwalk, CT) using the following conditions: denaturation (94°C, tion employing retroviral gene traps, introduced into cul- 30 s), annealing (58°C, 45 s), and extension (72°C, l min) for 35 cycles. tured mouse C2C12 mouse myoblasts. When C2C12 myo- TaqStart antibody (Clontech, Palo Alto, CA) was used to facilitate "hot start" PCR. 5 Ixl from the first round product were cut separately with 5 U blasts, growing in serum rich media, are placed into media of the following enzymes: XhoI or BamHl or Pstl that cut within the am- with low serum concentration, they undergo differentia- plified fragment originating from the gene trap vector. 1 Ixl of each one of tion characterized by the formation of myotubes. We have these digestions was then used in the second round of nested priming, us- been able to screen hundreds of genes differentially regu- ing primers C (CGCGTCGACCTrGCCAACCTACAGGT) and D (CTC- GCTTCTGTTCGCG) and conditions identical with the ones described lated during muscle maturation and selectively pursue the above except for 25 cycles. The size of the amplification products obtained cloning of those in which mutations produce the most in- was between 300 and 1,000 bp and on the average ,-~400 bp. A source of teresting phenotypes and/or patterns of expression with background is amplification products originating from endogenous retro- respect to temporal sequence and subcellular localization. viruses. These products co-appear occasionally (but not reproducibly) as Among the first genes that we have identified in this fragments of °'300 bp and can be easily recognized and discarded by se- quence analysis. The PCR product was subcloned in the TA vector (Invi- manner is the lysosomal cysteine protease cathepsin B. We trogen, San Diego, CA) and sequenced. find that its expression is induced in myoblasts by serum Sequence data from the products of the 5' RACE, inverse PCR, and starvation but downregulated in myotubes. Cells in which genomic walking was searched by BLAST ([email protected]). one cathepsin B allele is interrupted by the gene trap have a unique phenotype consisting of deficiency of myoblast Sequence Confirmation of Cathepsin B Integration fusion and an unusual growth morphology with decreased In the clone trapped at the cathepsin B locus, endogenous flanking se- postmitotic survival. The phenotype is reproducible in pa- quences were recovered through inverse PCR. Approximately 400 bp of rental C2C12 myoblasts with antisense cathepsin B ex- sequence downstream of the retroviral integration site from the 3' LTR of pression. These results implicate cathepsin B in myoblast the trapping vector to the leader C of mouse cathepsin B was essentially identical to published sequence data (Rhaissi et al., 1993). The integrity of growth and fusion and suggest that a specific deficiency of the upstream cathepsin B genomic sequence was confirmed by genomic cathepsin B could account for the myopathy of I cell dis- PCR, using a downstream primer contained within the I?,geo gene and ease, in which there is reduced localization of lysosomal three separate upstream primers, corresponding to genomic sequences enzymes. 1397 to 1420, 1610 to 1630, and 2329 to 2349. In each case, the expected size DNA fragment was uniquely amplified.
Load more

Recommended publications
  • Chapter 14: Functional Genomics Learning Objectives
    Chapter 14: Functional Genomics Learning objectives Upon reading this chapter, you should be able to: ■ define functional genomics; ■ describe the key features of eight model organisms; ■ explain techniques of forward and reverse genetics; ■ discuss the relation between the central dogma and functional genomics; and ■ describe proteomics-based approaches to functional genomics. Outline : Functional genomics Introduction Relation between genotype and phenotype Eight model organisms E. coli; yeast; Arabidopsis; C. elegans; Drosophila; zebrafish; mouse; human Functional genomics using reverse and forward genetics Reverse genetics: mouse knockouts; yeast; gene trapping; insertional mutatgenesis; gene silencing Forward genetics: chemical mutagenesis Functional genomics and the central dogma Approaches to function; Functional genomics and DNA; …and RNA; …and protein Proteomic approaches to functional genomics CASP; protein-protein interactions; protein networks Perspective Albert Blakeslee (1874–1954) studied the effect of altered chromosome numbers on the phenotype of the jimson-weed Datura stramonium, a flowering plant. Introduction: Functional genomics Functional genomics is the genome-wide study of the function of DNA (including both genes and non-genic regions), as well as RNA and proteins encoded by DNA. The term “functional genomics” may apply to • the genome, transcriptome, or proteome • the use of high-throughput screens • the perturbation of gene function • the complex relationship of genotype and phenotype Functional genomics approaches to high throughput analyses Relationship between genotype and phenotype The genotype of an individual consists of the DNA that comprises the organism. The phenotype is the outward manifestation in terms of properties such as size, shape, movement, and physiology. We can consider the phenotype of a cell (e.g., a precursor cell may develop into a brain cell or liver cell) or the phenotype of an organism (e.g., a person may have a disease phenotype such as sickle‐cell anemia).
    [Show full text]
  • Expression Profiling with Arrays of Randomly Disrupted Genes in Mouse Embryonic Stem Cells Leads to in Vivo Functional Analysis
    Expression profiling with arrays of randomly disrupted genes in mouse embryonic stem cells leads to in vivo functional analysis Eishou Matsuda*†, Toshiaki Shigeoka*†, Ryuji Iida*, Shinya Yamanaka‡, Masashi Kawaichi*, and Yasumasa Ishida*§ *Division of Gene Function in Animals, Graduate School of Biological Sciences, and ‡Laboratory of Animal Molecular Technology, Research and Education Center for Genetic Information, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma-shi, Nara 630-0192, Japan Communicated by Tasuku Honjo, Kyoto University, Kyoto, Japan, January 27, 2004 (received for review December 17, 2003) DNA arrays are capable of profiling the expression patterns of the target cells (8, 9). Although the promoter͞enhancer trap is many genes in a single experiment. After finding a gene of interest a very reliable way to focus exclusively on functional genes, in a DNA array, however, labor-intensive gene-targeting experi- transcriptionally silent genes in the target cells are missed by this ments sometimes must be performed for the in vivo analysis of the strategy. gene function. With random gene trapping, on the other hand, it Another way to select for intragenic vector integration is to is relatively easy to disrupt and retrieve hundreds of genes͞gene employ a polyadenylation [poly(A)] trap. In this case, an mRNA candidates in mouse embryonic stem (ES) cells, but one could transcribed from a selectable marker gene lacking a poly(A) overlook potentially important gene-disruption events if only the signal in a gene trap vector is stabilized only when the gene trap nucleotide sequences and not the expression patterns of the vector captures a cellular poly(A) signal (6, 10–13).
    [Show full text]
  • Constitutive Gs Activation Using a Single-Construct Tetracycline-Inducible Expression System in Embryonic Stem Cells and Mice
    UCSF UC San Francisco Previously Published Works Title Constitutive Gs activation using a single-construct tetracycline-inducible expression system in embryonic stem cells and mice Permalink https://escholarship.org/uc/item/6762x12t Journal Stem Cell Research & Therapy, 2(2) ISSN 1757-6512 Authors Hsiao, Edward C Nguyen, Trieu D Ng, Jennifer K et al. Publication Date 2011-03-04 DOI http://dx.doi.org/10.1186/scrt52 Supplemental Material https://escholarship.org/uc/item/6762x12t#supplemental Peer reviewed eScholarship.org Powered by the California Digital Library University of California Hsiao et al. Stem Cell Research & Therapy 2011, 2:11 http://stemcellres.com/content/2/2/11 RESEARCH Open Access Constitutive Gs activation using a single-construct tetracycline-inducible expression system in embryonic stem cells and mice Edward C Hsiao1,2*, Trieu D Nguyen1, Jennifer K Ng1, Mark J Scott1, Wei Chun Chang3, Hengameh Zahed4,5, Bruce R Conklin1,6 Abstract Introduction: The controlled expression of many genes, including G-protein coupled receptors (GPCRs), is important for delineating gene functions in complex model systems. Binary systems for inducible regulation of transgene expression are widely used in mice. One system is the tTA/TRE expression system, composed of a tetracycline-dependent DNA binding factor and a separate tetracycline operon. However, the requirement for two separate transgenes (one for each tTA or TRE component) makes this system less amenable to models requiring directed cell targeting, increases the risk of multiple transgene integration sites, and requires extensive screening for appropriately-functioning clones. Methods: We developed a single, polycistronic tetracycline-inducible expression platform to control the expression of multiple cistrons in mammalian cells.
    [Show full text]
  • BMC Biotechnology Biomed Central
    BMC Biotechnology BioMed Central Research article Open Access Conditional gene expression in the mouse using a Sleeping Beauty gene-trap transposon AronMGeurts*1, Andrew Wilber1,2, Corey M Carlson3, Paul D Lobitz1, Karl J Clark1, Perry B Hackett1,2,3, R Scott McIvor1,2,3 and David A Largaespada1,2,3 Address: 1Department of Genetics, Cell Biology, and Development and The Arnold and Mabel Beckman Center for Transposon Research at the University of Minnesota, Minneapolis, MN 55455, USA, 2Institute of Human Genetics, University of Minnesota, Minneapolis, MN 55455, USA and 3University of Minnesota Cancer Center, Minneapolis, MN 55455, USA Email: Aron M Geurts* - [email protected]; Andrew Wilber - [email protected]; Corey M Carlson - [email protected]; Paul D Lobitz - [email protected]; Karl J Clark - [email protected]; Perry B Hackett - [email protected]; R Scott McIvor - [email protected]; David A Largaespada - [email protected] * Corresponding author Published: 26 June 2006 Received: 08 March 2006 Accepted: 26 June 2006 BMC Biotechnology 2006, 6:30 doi:10.1186/1472-6750-6-30 This article is available from: http://www.biomedcentral.com/1472-6750/6/30 © 2006 Geurts et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Insertional mutagenesis techniques with transposable elements have been popular among geneticists studying model organisms from E. coli to Drosophila and, more recently, the mouse.
    [Show full text]
  • Generation of Hypoxanthine Phosphoribosyltransferase
    www.nature.com/scientificreports OPEN Generation of hypoxanthine phosphoribosyltransferase gene knockout rabbits by homologous Received: 09 March 2015 Accepted: 08 October 2015 recombination and gene trapping Published: 02 November 2015 through somatic cell nuclear transfer Mingru Yin*, Weihua Jiang*, Zhenfu Fang, Pengcheng Kong, Fengying Xing, Yao Li, Xuejin Chen & Shangang Li The rabbit is a common animal model that has been employed in studies on various human disorders, and the generation of genetically modified rabbit lines is highly desirable. Female rabbits have been successfully cloned from cumulus cells, and the somatic cell nuclear transfer (SCNT) technology is well established. The present study generated hypoxanthine phosphoribosyltransferase (HPRT) gene knockout rabbits using recombinant adeno-associated virus-mediated homologous recombination and SCNT. Gene trap strategies were employed to enhance the gene targeting rates. The male and female gene knockout fibroblast cell lines were derived by different strategies. When male HPRT knockout cells were used for SCNT, no live rabbits were obtained. However, when female HPRT+/− cells were used for SCNT, live, healthy rabbits were generated. The cloned HPRT+/− rabbits were fertile at maturity. We demonstrate a new technique to produce gene-targeted rabbits. This approach may also be used in the genetic manipulation of different genes or in other species. Animals with specific genetic mutations are powerful tools for investigating gene function and human diseases. Various genetic alterations have been induced in mice to improve our understanding of both human disease pathogenesis and the development of new treatment regimens. However, mice fail to replicate the phenotype of several diseases, including Lesch-Nyhan syndrome1,2 and cystic fibrosis3.
    [Show full text]
  • Jarid2 Is a PRC2 Component in Embryonic Stem Cells Required for Multi-Lineage Differentiation and Recruitment of PRC1 and RNA Po
    Europe PMC Funders Group Author Manuscript Nat Cell Biol. Author manuscript; available in PMC 2013 February 14. Published in final edited form as: Nat Cell Biol. 2010 June ; 12(6): 618–624. doi:10.1038/ncb2065. Europe PMC Funders Author Manuscripts Jarid2 is a PRC2 component in embryonic stem cells required for multi-lineage differentiation and recruitment of PRC1 and RNA Polymerase II to developmental regulators David Landeira1,8, Stephan Sauer1,8, Raymond Poot2, Maria Dvorkina1, Luca Mazzarella1, Helle F. Jørgensen1, C. Filipe Pereira1, Marion Leleu1, Francesco M. Piccolo1, Mikhail Spivakov1, Emily Brookes3, Ana Pombo3, Cynthia Fisher1,4, William C. Skarnes4, Tim Snoek2, Karel Bezstarosti5, Jeroen Demmers5, Robert J. Klose6, Miguel Casanova7, Ligia Tavares7, Neil Brockdorff7, Matthias Merkenschlager1, and Amanda G. Fisher1,9 1Lymphocyte Development Group, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN UK 2Department of Cell Biology, Erasmus Medical Centre, Dr. Molewaterplein 50, 3015GE, Rotterdam, Netherlands 3Genome Function Group, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN UK 4Mouse Developmental Genetics Group, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA UK 5Proteomics Center, Erasmus Medical Centre, Dr. Molewaterplein 50, 3015GE, Rotterdam, Netherlands 6Epigenetic Regulation of Chromatin Function Group Department of Biochemistry,
    [Show full text]
  • A Conditional Knockout Resource for the Genome-Wide Study of Mouse Gene Function
    ARTICLE doi:10.1038/nature10163 A conditional knockout resource for the genome-wide study of mouse gene function William C. Skarnes1, Barry Rosen1, Anthony P. West1, Manousos Koutsourakis1, Wendy Bushell1, Vivek Iyer1, Alejandro O. Mujica1{, Mark Thomas1, Jennifer Harrow1, Tony Cox1, David Jackson1, Jessica Severin1{, Patrick Biggs1{, Jun Fu2, Michael Nefedov3, Pieter J. de Jong3, A. Francis Stewart2 & Allan Bradley1 Gene targeting in embryonic stem cells has become the principal technology for manipulation of the mouse genome, offering unrivalled accuracy in allele design and access to conditional mutagenesis. To bring these advantages to the wider research community, large-scale mouse knockout programmes are producing a permanent resource of targeted mutations in all protein-coding genes. Here we report the establishment of a high-throughput gene-targeting pipeline for the generation of reporter-tagged, conditional alleles. Computational allele design, 96-well modular vector construction and high-efficiency gene-targeting strategies have been combined to mutate genes on an unprecedented scale. So far, more than 12,000 vectors and 9,000 conditional targeted alleles have been produced in highly germline-competent C57BL/6N embryonic stem cells. High-throughput genome engineering highlighted by this study is broadly applicable to rat and human stem cells and provides a foundation for future genome-wide efforts aimed at deciphering the function of all genes encoded by the mammalian genome. Following the complete sequencing of the human and mouse genomes, and termination signals of each target gene, thus requiring no previous the functional analysis of each of the twenty thousand or so protein- knowledge of the underlying genomic structure of the gene.
    [Show full text]
  • Gene-Trap Mutagenesis: Past, Present and Beyond
    REVIEWS GENE-TRAP MUTAGENESIS: PAST, PRESENT AND BEYOND William L. Stanford*, Jason B. Cohn*‡ and Sabine P. Cordes*‡ Although at least 35,000 human genes have been sequenced and mapped, adequate expression or functional information is available for only ~15% of them. Gene-trap mutagenesis is a technique that randomly generates loss-of-function mutations and reports the expression of many mouse genes. At present, several large-scale, gene-trap screens are being carried out with various new vectors, which aim to generate a public resource of mutagenized embryonic stem (ES) cells. This resource now includes more than 8,000 mutagenized ES-cell lines, which are freely available, making it an appropriate time to evaluate the recent advances in this area of genomic technology and the technical hurdles it has yet to overcome. MOUSE GENOMIC TECHNOLOGIES INTRACISTERNAL A PARTICLE (IAP). Endogenous, non- In Simone de Beauvoir’s existentialist novel All Men Are low frequency at which spontaneous mutations occur infectious retroviral element Mortal, two creatures attain immortality: a man who (~5 × 10−6 per locus) makes dissecting an entire genetic that can undergo transposition intends to be an enlightened ruler and a circling mouse. pathway by awaiting the appearance of spontaneous and act as a mutagen. Clearly, this was a mutant mouse — perhaps a descen- mutations unfeasible in a normal human lifespan or ALLELIC SERIES dant of the Chinese waltzing mice that were first with the resources of an averagely sized mouse house. In A collection of discrete described several thousand years ago. de Beauvoir’s addition, the molecular lesions that are responsible for mutations that affect the same would-be king chose the mouse as his companion for spontaneous mutations can range from small base gene.
    [Show full text]
  • Two Ways to Trap a Gene in Mice
    COMMENTARY Two ways to trap a gene in mice William C. Skarnes* Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, United Kingdom he sequencing of the human tion of random insertional mutations in and mouse genomes has been ES cells (13). Both privately and pub- heralded as one of the most licly funded efforts have generated large important recent achievements libraries of ES cells harboring gene-trap Tin biological science. Attention has now insertions (the International Gene Trap turned to the functional annotation of Consortium maintains a database of the 25,000 or so genes encoded by the gene-trap cell lines available from the mammalian genome. Although there are public resource at www.genetrap.org). many experimental approaches that ad- Combined, these resources are estimated dress gene function, the most relevant to contain mutations in Ͼ60% of genes approach for extrapolation to human in mice (14). Gene-trap libraries have development, physiology, and disease is become an important source of new mu- to analyze the phenotype of mutations tations in mice for the pharmaceutical for each gene in a whole mammalian industry and academic investigators. model, the mouse. Many phenotypes The National Institutes of Health- relevant to congenital traits and abnor- funded BayGenomics program (http:͞͞ mal pathologies in humans have baygenomics.ucsf.edu), for example, has emerged from gene knockout studies in distributed Ͼ2,000 gene-trap ES cell the mouse, providing a strong justifica- lines to academic laboratories. Clearly, tion for expanding the collection of there is a high demand for mutant ES mouse mutants to include all genes.
    [Show full text]
  • A Versatile Gene Trap to Visualize and Interrogate the Function of the Vertebrate Proteome
    View metadata, citation andDownloaded similar papers from at genesdev.cshlp.org core.ac.uk on December 31, 2015 - Published by Cold Spring Harbor Laboratory Press brought to you by CORE provided by Caltech Authors RESOURCE/METHODOLOGY A versatile gene trap to visualize and interrogate the function of the vertebrate proteome Le A. Trinh,1,5 Tatiana Hochgreb,1 Matthew Graham,2 David Wu,1 Frederique Ruf-Zamojski,1,3 Chathurani S. Jayasena,1 Ankur Saxena,1 Rasheeda Hawk,1 Aidyl Gonzalez-Serricchio,1 Alana Dixson,1 Elly Chow,1 Constanza Gonzales,1 Ho-Yin Leung,1 Ilana Solomon,1 Marianne Bronner-Fraser,1 Sean G. Megason,1,4 and Scott E. Fraser1 1Beckman Institute, Division of Biology, and 2Center for Advanced Computing Research, California Institute of Technology, Pasadena, California 91125, USA We report a multifunctional gene-trapping approach, which generates full-length Citrine fusions with endogenous proteins and conditional mutants from a single integration event of the FlipTrap vector. We identified 170 FlipTrap zebrafish lines with diverse tissue-specific expression patterns and distinct subcellular localizations of fusion proteins generated by the integration of an internal citrine exon. Cre-mediated conditional mutagenesis is enabled by heterotypic lox sites that delete Citrine and ‘‘flip’’ in its place mCherry with a polyadenylation signal, resulting in a truncated fusion protein. Inducing recombination with Cerulean-Cre results in fusion proteins that often mislocalize, exhibit mutant phenotypes, and dramatically knock down wild-type transcript levels. FRT sites in the vector enable targeted genetic manipulation of the trapped loci in the presence of Flp recombinase. Thus, the FlipTrap captures the functional proteome, enabling the visualization of full-length fluorescent fusion proteins and interrogation of function by conditional mutagenesis and targeted genetic manipulation.
    [Show full text]
  • A Gene-Trap Strategy Identifies Quiescence-Induced Genes
    Gene trapping in reversibly arrested myoblasts 27 A gene-trap strategy identifi es quiescence-induced genes in synchronized myoblasts RAMKUMAR SAMBASIVAN1,3, GRACE K PAVLATH2 and JYOTSNA DHAWAN1 1Centre for Cellular and Molecular Biology, Hyderabad 500 007, India 2Department of Pharmacology, Emory University, Atlanta, GA 30322, USA 3Present address: Department of Developmental Biology, Pasteur Institute, 75724 Cedex 15 Paris, France *Corresponding author (Fax, 91-40-2716-0591; Email, [email protected]) Cellular quiescence is characterized not only by reduced mitotic and metabolic activity but also by altered gene expression. Growing evidence suggests that quiescence is not merely a basal state but is regulated by active mechanisms. To understand the molecular programme that governs reversible cell cycle exit, we focused on quiescence-related gene expression in a culture model of myogenic cell arrest and activation. Here we report the identifi cation of quiescence- induced genes using a gene-trap strategy. Using a retroviral vector, we generated a library of gene traps in C2C12 myoblasts that were screened for arrest-induced insertions by live cell sorting (FACS-gal). Several independent gene- trap lines revealed arrest-dependent induction of βgal activity, confi rming the effi cacy of the FACS screen. The locus of integration was identifi ed in 15 lines. In three lines, insertion occurred in genes previously implicated in the control of quiescence, i.e. EMSY – a BRCA2-interacting protein, p8/com1– a p300HAT-binding protein and MLL5 – a SET domain protein. Our results demonstrate that expression of chromatin modulatory genes is induced in G0, providing support to the notion that this reversibly arrested state is actively regulated.
    [Show full text]
  • A Review of Current Large-Scale Mouse Knockout Efforts
    ' 2010 Wiley-Liss, Inc. genesis 48:73–85 (2010) REVIEW A Review of Current Large-Scale Mouse Knockout Efforts Chunmei Guan, Chao Ye, Xiaomei Yang,* and Jiangang Gao* College of Life Science, Shandong University, Jinan 250100, Shandong, People’s Republic of China Received 27 August 2009; Revised 25 October 2009; Accepted 13 November 2009 Summary: After the successful completion of the human 2003; Collins and Mansoura, 2001; Gottesman and genome project (HGP), biological research in the post- Collins, 1994). Biological research has entered a new genome era urgently needs an efficient approach for stage—the post-genome era, and the main work of this functional analysis of genes. Utilization of knockout era is to decipher the function of each gene in our ge- mouse models has been powerful for elucidating the nome (Austin et al., 2004; Eisenberg et al., 2000). Com- function of genes as well as finding new therapeutic interventions for human diseases. Gene trapping and parative genomics has shown that the mouse and human gene targeting are two independent techniques for mak- genomes have high homology (Gregory et al., 2002). ing knockout mice from embryonic stem (ES) cells. Gene Therefore, the mouse serves as a perfect model animal trapping is high-throughput, random, and sequence- for functional genomic research of humans. The most ef- tagged while gene targeting enables the knockout of ficient way to study the function of a gene is to make a specific genes. It has been about 20 years since the first knockout and observe the phenotype in the whole ani- gene targeting and gene trapping mice were generated.
    [Show full text]