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Harald Von Melchner - One of the best experts on this subject based on the ideXlab platform.

  • Generation of a multipurpose prdm16 mouse allele by targeted Gene Trapping
    Disease Models & Mechanisms, 2017
    Co-Authors: Alexander Strassman, Frank Schnutgen, Qi Dai, Jennifer C Jones, Angela C Gomez, Lenore Pitstick, Nathan E Holton, Russell Moskal, Erin R Leslie, Harald Von Melchner
    Abstract:

    Gene trap mutaGenesis is a powerful tool to create loss-of-function mutations in mice and other model organisms. Modifications of traditional Gene trap cassettes, including addition of conditional features in the form of Flip-excision (FlEx) arrays to enable directional Gene trap cassette inversions by Cre and Flpe site-specific recombinases, greatly enhanced their experimental potential. By taking advantage of these conditional Gene trap cassettes, we developed a Generic strategy for Generating conditional mutations and validated this strategy in mice carrying a multipurpose allele of the Prdm16 transcription factor Gene. We demonstrate that the Gene trap insertion creates a null mutation replicating the Pierre Robin sequence-type cleft palate phenotype of other Prdm16 mutant mice. Consecutive breeding to Flpe and Emx1IREScre deleter mice spatially restricted Prdm16 loss to regions of the forebrain expressing the homeobox Gene Emx1, demonstrating the utility of the technology for the analysis of tissue-specific Gene functions.

  • high throughput Gene Trapping and postinsertional modifications of Gene trap alleles
    Methods, 2011
    Co-Authors: Frank Schnutgen, Franziska Ehrmann, Patricia Ruiznoppinger, Harald Von Melchner
    Abstract:

    Gene Trapping is a high-throughput insertional mutaGenesis approach that has been primarily used in mouse embryonic stem cells (ESCs). As a high throughput technology, Gene Trapping helped to Generate tenth of thousands of ESC lines harboring mutations in single Genes that can be used for making knock-out mice. Ongoing international efforts operating under the umbrella of the International Knockout Mouse Consortium (IKMC; www.knockoutmouse.org) aim to Generate conditional alleles for every protein coding Gene in the mouse genome by high throughput conditional Gene targeting and Trapping. Here, we provide protocols for Gene Trapping in ESCs that can be easily adapted to any other mammalian cell. We further provide protocols for handling and verifying conditional Gene trap alleles in ESC lines obtained from the IKMC repositories and describe a highly efficient method for the postinsertional modification of Gene trap alleles. More specifically, we describe a protein tagging strategy based on recombinase mediated cassette exchange (RMCE) that enables protein localization and protein-protein interaction studies under physiological conditions.

  • Gene Trapping identifies a putative tumor suppressor and a new inducer of cell migration
    Biochemical and Biophysical Research Communications, 2008
    Co-Authors: Francisca Guardiolaserrano, Harald Von Melchner, Judith Haendeler, Margarete Lukosz, Karsten Sturm, Joachim Altschmied
    Abstract:

    Abstract Tumor necrosis factor alpha (TNFα) is a pleiotropic cytokine involved in apoptotic cell death, cellular proliferation, differentiation, inflammation, and tumoriGenesis. In tumors it is secreted by tumor associated macrophages and can have both pro- and anti-tumorigenic effects. To identify Genes regulated by TNFα, we performed a Gene trap screen in the mammary carcinoma cell line MCF-7 and recovered 64 unique, TNFα-induced Gene trap integration sites. Among these were the Genes coding for the zinc finger protein ZC3H10 and for the transcription factor grainyhead-like 3 (GRHL3). In line with the dual effects of TNFα on tumoriGenesis, we found that ZC3H10 inhibits anchorage independent growth in soft agar suggesting a tumor suppressor function, whereas GRHL3 strongly stimulated the migration of endothelial cells which is consistent with an angiogenic, pro-tumorigenic function.

  • enhanced Gene Trapping in mouse embryonic stem cells
    Nucleic Acids Research, 2008
    Co-Authors: Frank Schnutgen, Jens Hansen, Silke Dezolt, Carsten Horn, Marcus Lutz, Thomas Floss, Wolfgang Wurst, Patricia Ruiz Noppinger, Harald Von Melchner
    Abstract:

    Gene Trapping is used to introduce insertional mutations into Genes of mouse embryonic stem cells (ESCs). It is performed with Gene trap vectors that simultaneously mutate and report the expression of the endogenous Gene at the site of insertion and provide a DNA tag for rapid identification of the disrupted Gene. Gene traps have been employed worldwide to assemble libraries of mouse ESC lines harboring mutations in single Genes, which can be used to make mutant mice. However, most of the employed Gene trap vectors require Gene expression for reporting a Gene trap event and therefore Genes that are poorly expressed may be under-represented in the existing libraries. To address this problem, we have developed a novel class of Gene trap vectors that can induce Gene expression at insertion sites, thereby bypassing the problem of intrinsic poor expression. We show here that the insertion of the osteopontin enhancer into several conventional Gene trap vectors significantly increases the Gene Trapping efficiency in high-throughput screens and facilitates the recovery of poorly expressed Genes.

  • a public Gene trap resource for mouse functional genomics
    Nature Genetics, 2004
    Co-Authors: William C Skarnes, Harald Von Melchner, Wolfgang Wurst, Geoff Hicks, Alexander Nord, Tony Cox, Stephen G Young, Patricia Ruiz, Phil Soriano, Marc Tessierlavigne
    Abstract:

    To the editor: Gene Trapping is a high-throughput approach that can be used to introduce insertional mutations across the genome in mouse embryonic stem (ES) cells. Gene trap vectors simultaneously mutate and report the expression of the endogenous Gene at the site of insertion and provide a DNA tag for the rapid identification of the disrupted Gene. The Generation of mutant mice from a large collection of ES cell lines carrying Gene trap insertions could be applied to large-scale functional analysis of the ~30,000 mammalian Genes. The overall impact of Gene trap resources will rest on the fraction of the genome that is accessible with this technology, the efficiency relative to other competing technologies and the availability of such a resource to the academic community. Lexicon Genetics, a US-based biotechnology company, was the first to implement a genome-wide Gene Trapping program1 and has developed OmniBank (http://www.lexicon-Genetics.com), the largest library of mutant ES cell lines. A parallel effort was initiated in the public domain by several academic groups in the International Gene Trap Consortium (IGTC; http://www.igtc.ca). The recent release of OmniBank sequence tags to GenBank2 has made it possible to compare the size and efficiency of the existing Gene trap libraries. We confirm that Lexicon achieved close to 60% coverage of the genome from 200,000 OmniBank sequence tags deposited in GenBank (Fig. 1). Our analysis, supported independently by Lexicon3, indicates that the rate of Trapping new Genes was not linear but declined within the first 100,000 tags to a rate at which 1 new Gene was added every 35 tags, comparable to the efficiency of high-throughput Gene targeting methods4. To date, the IGTC has attained 32% genome coverage in 27,000 tags; Trapping is likewise nonlinear, but the initial rate seems to be somewhat faster than Lexicon’s (Fig. 1). The seemingly higher efficiency may relate to the diversity of plasmid and retroviral vector designs used by the IGTC that could help overcome insertion site preferences of any single vector5; further studies are needed to fully understand how vector design and other experimental factors influence the efficiency of Gene Trapping. One-fifth of the Genes trapped by the IGTC were not represented in the sequence tags released by Lexicon (Supplementary Tables 1-3 online). Thus, the two efforts together have trapped nearly two-thirds of all Genes in mice. We conclude that Gene Trapping is an effective strategy to mutate a substantial fraction of the Genes in mice that compares favorably with Gene-targeting approaches. Furthermore, we continue to refine the technology, particularly in developing strategies for postinsertional modification of the trapped loci to create a wide range of desired alleles. The IGTC will provide an important public resource of new mutations in mice that will accelerate the pace of functional annotation of the mammalian genome. Figure 1 Comparison of the rates of Trapping of the IGTC and OmniBank resources. Unique Ensembl Genes were identified using MAPTAG (http://www.sanger.ac.uk/Software/MAPTAG), an automated annotation program that identifies short, almost perfect matches to Gene ... Gene trap cell lines Generated by the IGTC are available without restriction (http://baygenomics.ucsf.edu; http://www.Genetrap.de; http://www.escells.ca; http://www.sanger.ac.uk/Genetrap; http://www.fhcrc.org/labs/soriano/GTdb; http://www.cmhd.ca) and all sequence tags are mapped on the Ensembl mouse genome browser http://www.ensembl.org/Mus_musculus/; select DAS Source ‘GeneTrap’).

Koichi Kawakami - One of the best experts on this subject based on the ideXlab platform.

  • Tol2-mediated TransGenesis, Gene Trapping, Enhancer Trapping, and the Gal4-UAS System
    Methods in cell biology, 2016
    Co-Authors: Koichi Kawakami, Kazuhide Asakawa, Akira Muto, Hironori Wada
    Abstract:

    The Tol2 transposable element was originally found in the genome of the Japanese medaka fish (Oryzias latipes). Tol2 contains a Gene encoding an active transposase that can catalyze DNA transposition in vertebrate cells. In zebrafish, Tol2 Generates genomic integrations in the germ cells very efficiently. By using the Tol2 transposition system, we have developed important Genetic methods including transGenesis, Gene Trapping, enhancer Trapping, and the Gal4-UAS system in zebrafish. In this chapter, we describe how these methods can be performed.

  • cellular dissection of the spinal cord motor column by bac transGenesis and Gene Trapping in zebrafish
    Frontiers in Neural Circuits, 2013
    Co-Authors: Koichi Kawakami, Kazuhide Asakawa, Gembu Abe
    Abstract:

    Bacterial artificial chromosome (BAC) transGenesis and Gene/enhancer Trapping are effective approaches for identification of Genetically defined neuronal populations in the central nervous system (CNS). Here, we applied these techniques to zebrafish (Danio rerio) in order to obtain insights into the cellular architecture of the axial motor column in vertebrates. First, by using the BAC for the Mnx class homeodomain protein Gene mnr2b/mnx2b, we established the mnGFF7 transgenic line expressing the Gal4FF transcriptional activator in a large part of the motor column. Single cell labeling of Gal4FF-expressing cells in the mnGFF7 line enabled a detailed investigation of the morphological characteristics of individual spinal motoneurons, as well as the overall organization of the motor column in a spinal segment. Secondly, from a large-scale Gene trap screen, we identified transgenic lines that marked discrete subpopulations of spinal motoneurons with Gal4FF. Molecular characterization of these lines led to the identification of the ADAMTS3 Gene, which encodes an evolutionarily conserved ADAMTS family of peptidases and is dynamically expressed in the ventral spinal cord. The transgenic fish established here, along with the identified Gene, should facilitate an understanding of the cellular and molecular architecture of the spinal cord motor column and its connection to muscles in vertebrates.

  • efficient transposition of the tol2 transposable element from a single copy donor in zebrafish
    Proceedings of the National Academy of Sciences of the United States of America, 2008
    Co-Authors: Akihiro Urasaki, Kazuhide Asakawa, Koichi Kawakami
    Abstract:

    The Tol2 transposable element is a powerful Genetic tool in model vertebrates and has been used for transGenesis, insertional mutaGenesis, Gene Trapping, and enhancer Trapping. However, an in vivo transposition system using Tol2 has not yet been developed. Here we report the in vivo Tol2 transposition system in a model vertebrate, zebrafish. First, we constructed transgenic zebrafish that carried single-copy integrations of Tol2 on the genome and injected transposase mRNA into one-cell stage embryos. The Tol2 insertions were mobilized efficiently in the germ lineage. We then mobilized an insertion of the Tol2 Gene trap construct in the nup214 Gene, which caused a recessive lethal mutant phenotype, and demonstrated that this method is applicable to the isolation of revertants from a transposon insertional mutant. Second, we constructed transgenic fish carrying the transposase cDNA under the control of the hsp70 promoter. Double-transgenic fish containing the transposase Gene and a single-copy Tol2 insertion were treated with heat shock at the adult stage. We found that transposition can be induced efficiently in the male germ cells. We analyzed new integration sites and found that the majority (83%) of them were mapped on chromosomes other than the transposon donor chromosomes and that 9% of local hopping events mapped less than 300 kb away from the donor loci. Our present study demonstrates that the in vivo Tol2 transposition system is useful for creating genome-wide insertions from a single-copy donor and should facilitate functional genomics and transposon biology in vertebrates.

  • transposon mediated Gene Trapping in zebrafish
    Methods, 2006
    Co-Authors: Tomoya Kotani, Akihiro Urasaki, Saori Nagayoshi, Koichi Kawakami
    Abstract:

    The Tol2 transposon system can create chromosomal insertions in the zebrafish germ lineage very efficiently. We constructed a Tol2-based Gene trap vector, T2KSAG, which contains a splice accepter, the GFP Gene and the polyA signal. In the pilot screen for Gene Trapping using T2KSAG, we identified 38 fish lines expressing GFP in specific organs and tissues. In the SAGp53A line, GFP is expressed in the forebrain and midbrain, and the insertion of the Gene trap construct captured a transcript of the kab Gene encoding a zebrafish homolog of the human KARP (Ku86 autoantigen related protein)-binding protein (KAB). In the SAGm18B line, GFP is expressed in the central nervous system, and the insertion captured a transcript of a Gene for succinyl CoA:3-oxoacid CoA-transferase (SCOT). Here, we describe how we performed the Gene trap screen and characterized the Gene trap insertions and will discuss the outcome of the pilot screen.

  • a transposon mediated Gene trap approach identifies developmentally regulated Genes in zebrafish
    Developmental Cell, 2004
    Co-Authors: Koichi Kawakami, Hisashi Takeda, Noriko Kawakami, Makoto Kobayashi, Naoto Matsuda, Masayoshi Mishina
    Abstract:

    We report here development of a novel Gene trap method in zebrafish using the Tol2 transposon system. First, we established a highly efficient transGenesis method in which a plasmid DNA containing the Tol2 transposon vector and the transposase mRNA synthesized in vitro were coinjected into one-cell stage embryos. The transposon vector inserted in the genome could be transmitted to the F1 progeny at high frequencies, and regulated Gene expression by a specific promoter could be recapitulated in transgenic fish. Then we constructed a transposon-based Gene trap vector containing a splice acceptor and the GFP Gene, performed a pilot screen for Gene Trapping, and obtained fish expressing GFP in temporally and spatially restricted patterns. We confirmed the endogenous transcripts were indeed trapped by the insertions, and the insertion could interfere with expression of the trapped Gene. We propose our Gene trap approach should facilitate studies of vertebrate development and organoGenesis.

William C Skarnes - One of the best experts on this subject based on the ideXlab platform.

  • a public Gene trap resource for mouse functional genomics
    Nature Genetics, 2004
    Co-Authors: William C Skarnes, Harald Von Melchner, Wolfgang Wurst, Geoff Hicks, Alexander Nord, Tony Cox, Stephen G Young, Patricia Ruiz, Phil Soriano, Marc Tessierlavigne
    Abstract:

    To the editor: Gene Trapping is a high-throughput approach that can be used to introduce insertional mutations across the genome in mouse embryonic stem (ES) cells. Gene trap vectors simultaneously mutate and report the expression of the endogenous Gene at the site of insertion and provide a DNA tag for the rapid identification of the disrupted Gene. The Generation of mutant mice from a large collection of ES cell lines carrying Gene trap insertions could be applied to large-scale functional analysis of the ~30,000 mammalian Genes. The overall impact of Gene trap resources will rest on the fraction of the genome that is accessible with this technology, the efficiency relative to other competing technologies and the availability of such a resource to the academic community. Lexicon Genetics, a US-based biotechnology company, was the first to implement a genome-wide Gene Trapping program1 and has developed OmniBank (http://www.lexicon-Genetics.com), the largest library of mutant ES cell lines. A parallel effort was initiated in the public domain by several academic groups in the International Gene Trap Consortium (IGTC; http://www.igtc.ca). The recent release of OmniBank sequence tags to GenBank2 has made it possible to compare the size and efficiency of the existing Gene trap libraries. We confirm that Lexicon achieved close to 60% coverage of the genome from 200,000 OmniBank sequence tags deposited in GenBank (Fig. 1). Our analysis, supported independently by Lexicon3, indicates that the rate of Trapping new Genes was not linear but declined within the first 100,000 tags to a rate at which 1 new Gene was added every 35 tags, comparable to the efficiency of high-throughput Gene targeting methods4. To date, the IGTC has attained 32% genome coverage in 27,000 tags; Trapping is likewise nonlinear, but the initial rate seems to be somewhat faster than Lexicon’s (Fig. 1). The seemingly higher efficiency may relate to the diversity of plasmid and retroviral vector designs used by the IGTC that could help overcome insertion site preferences of any single vector5; further studies are needed to fully understand how vector design and other experimental factors influence the efficiency of Gene Trapping. One-fifth of the Genes trapped by the IGTC were not represented in the sequence tags released by Lexicon (Supplementary Tables 1-3 online). Thus, the two efforts together have trapped nearly two-thirds of all Genes in mice. We conclude that Gene Trapping is an effective strategy to mutate a substantial fraction of the Genes in mice that compares favorably with Gene-targeting approaches. Furthermore, we continue to refine the technology, particularly in developing strategies for postinsertional modification of the trapped loci to create a wide range of desired alleles. The IGTC will provide an important public resource of new mutations in mice that will accelerate the pace of functional annotation of the mammalian genome. Figure 1 Comparison of the rates of Trapping of the IGTC and OmniBank resources. Unique Ensembl Genes were identified using MAPTAG (http://www.sanger.ac.uk/Software/MAPTAG), an automated annotation program that identifies short, almost perfect matches to Gene ... Gene trap cell lines Generated by the IGTC are available without restriction (http://baygenomics.ucsf.edu; http://www.Genetrap.de; http://www.escells.ca; http://www.sanger.ac.uk/Genetrap; http://www.fhcrc.org/labs/soriano/GTdb; http://www.cmhd.ca) and all sequence tags are mapped on the Ensembl mouse genome browser http://www.ensembl.org/Mus_musculus/; select DAS Source ‘GeneTrap’).

  • defining brain wiring patterns and mechanisms through Gene Trapping in mice
    Nature, 2001
    Co-Authors: Philip A Leighton, William C Skarnes, Kevin J Mitchell, Lisa V Goodrich, Kathy Pinson, Paul Scherz, Marc Tessierlavigne
    Abstract:

    The search to understand the mechanisms regulating brain wiring has relied on biochemical purification approaches in vertebrates and Genetic approaches in invertebrates to identify molecular cues and receptors for axon guidance. Here we describe a phenotype-based Gene-trap screen in mice designed for the large-scale identification of Genes controlling the formation of the trillions of connections in the mammalian brain. The method incorporates an axonal marker, which helps to identify cell-autonomous mechanisms in axon guidance, and has Generated a resource of mouse lines with striking patterns of axonal labelling, which facilitates analysis of the normal wiring diagram of the brain. Studies of two of these mouse lines have identified an in vivo guidance function for a vertebrate transmembrane semaphorin, Sema6A, and have helped re-evaluate that of the Eph receptor EphA4.

  • glypican 3 controls cellular responses to bmp4 in limb patterning and skeletal development
    Developmental Biology, 2000
    Co-Authors: Stephenie Painesaunders, Beth L Viviano, Joel Zupicich, William C Skarnes, Scott Saunders
    Abstract:

    Abstract Glypicans represent a family of six cell surface heparan sulfate proteoglycans in vertebrates. Although no specific in vivo functions have thus far been described for these proteoglycans, spontaneous mutations in the human and induced deletions in the mouse glypican-3 (Gpc3) Gene result in severe malformations and both pre- and postnatal overgrowth, known clinically as the Simpson–Golabi–Behmel syndrome (SGBS). Mice carrying mutant alleles of Gpc3 created by either targeted Gene disruption or Gene Trapping display a wide range of phenotypes associated with SGBS including renal cystic dysplasia, ventral wall defects, and skeletal abnormalities that are consistent with the pattern of Gpc3 expression in the mouse embryo. Previous studies in Drosophila have implicated glypicans in the signaling of decapentaplegic, a BMP homolog. Our experiments with mice show a significant relationship between vertebrate BMP signaling and glypican function; GPC3-deficient animals were mated with mice haploinsufficient for bone morphoGenetic protein-4 (Bmp4) and their offspring displayed a high penetrance of postaxial polydactyly and rib malformations not observed in either parent strain. This previously unknown link between glypican-3 and BMP4 function provides evidence of a role for glypicans in vertebrate limb patterning and skeletal development and suggests a mechanism for the skeletal defects seen in SGBS.

  • Gene Trapping methods for the identification and functional analysis of cell surface proteins in mice
    Methods in Enzymology, 2000
    Co-Authors: William C Skarnes
    Abstract:

    Publisher Summary Gene Trapping offers an alternative method to create random insertional mutations that are immediately accessible to molecular characterization. By eliminating the time-consuming step of constructing targeting vectors for each Gene of interest, the rate at which new mutations may be introduced in the germ line by Gene Trapping far exceeds that of conventional Gene targeting. Thus, Gene Trapping represents a rapid and cost-efficient method for the identification and functional analysis of new Genes in mice. Gene trap vectors are activated through the production of a reporter Gene fusion transcript following insertions of the vector within endogenous transcription units. Given the time and resources required for the phenotypic analysis of mutant mice, it is advantageous to tailor screens toward specific Genes of interest. A variety of criteria may be used to preselect insertional mutants prior to germ line transmission and phenotype analysis. These include the sequence of the target Gene, the expression profile of the target Gene, and the subcellular localization of the fusion product. This chapter focuses on a strategy developed to select insertional mutations specifically in Genes encoding cell surface proteins based on the subcellular localization of the fusion protein products in embryonic stem (ES) cells. An overview of this technology is presented in the chapter followed by a detailed description of the experimental procedures.

  • Gene Trapping to identify and analyze Genes expressed in the mouse hippocampus
    Hippocampus, 1998
    Co-Authors: Muriel Steel, William C Skarnes, Julie Moss, Katherine A Clark, Ian R Kearns, Ceri H Davies, Richard G M Morris, Richard Lathe
    Abstract:

    Mice harboring random Gene-trap insertions of a lacZ (b-galactosidase)-neomycin resistance fusion cassette (b-geo) were ana- lyzed for expression in the hippocampus. In 4 of 15 lines reporter Gene activity was observed in the hippocampal formation. In the obn line, enzyme activity was detected in the CA1-3 hippocampal subfields, in hpk expression was restricted to CA1, but in both lines reporter activity was also present in other brain regions. In the third line, kin, reporter activity was robustly expressed throughout the stratum pyrimidale of CA1-3, with only low-level expression elsewhere. The final line (glnC) displayed ubiquitous expression of the reporter and was not analyzed further. Fusion transcripts for the first three lines were characterized; all encode polypep- tides with features of membrane-associated signalling proteins. The obn fusion identified a human cDNA (B2-1) encoding a pleckstrin homology (PH) domain, while hpk sequences matched the Epstein-Barr Virus (EBV) inducible G-protein coupled receptor, EBI-1. kin identified an alternative form of the abl-related nonreceptor tyrosine kinase c-arg. Electrophysiologi- cal studies on mice homozygous for the insertions revealed normal synaptic transmission, paired pulse facilitation and paired-pulse depres- sion at Schaffer collateral-commissural CA1 synapses, and normal long- term potentiation (LTP) in obn and kin. hpk mice displayed an increase in hippocampal CA1 long-term potentiation (LTP), suggesting a role for this receptor in synaptic plasticity. Hippocampus 1998;8:444-457. r 1998 Wiley-Liss, Inc.

Alan Bernstein - One of the best experts on this subject based on the ideXlab platform.

  • craniofacial dysmorphoGenesis including cleft palate in mice with an insertional mutation in the discs large Gene
    Molecular and Cellular Biology, 2001
    Co-Authors: Georgina Caruana, Alan Bernstein
    Abstract:

    The discs large (Dlg) protein, or synapse-associated protein 97 (SAP97), is a member of the membrane-associated guanylate kinase family of multidomain scaffolding proteins which recruits transmembrane and signaling molecules to localized plasma membrane sites. Murine dlg is the homologue of the Drosophila dlg tumor suppressor Gene. The loss of dlg function in Drosophila disrupts cellular growth control, apicobasal polarity, and cell adhesion of imaginal disc epithelial cells, resulting in embryonic lethality. In this study, we isolated a mutational insertion in the murine dlg locus by Gene Trapping in totipotent embryonic stem cells. This insertion results in a truncated protein product that contains the N-terminal three PSD-95/DLG/ZO-1 domains of Dlg fused to the LacZ reporter and subsequently lacks the src homology 3 (SH3), protein 4.1 binding, and guanylate kinase (GUK)-like domains. The Dlg-LacZ fusion protein is expressed in epithelial, mesenchymal, neuronal, endothelial, and hematopoietic cells during embryoGenesis. Mice homozygous for the dlg mutation exhibit growth retardation in utero, have hypoplasia of the premaxilla and mandible, have a cleft secondary palate, and die perinatally. Consistent with this phenotype, Dlg-LacZ is expressed in mesenchymal and epithelial cells throughout palatal development. Our Genetic and phenotypic analysis of dlg mutant mice suggests that protein-protein interactions involving the SH3, protein 4.1 binding, and/or GUK-like domains are essential to the normal function of murine Dlg within craniofacial and palatal morphoGenesis.

  • Gene Trapping of two novel Genes hzf and hhl expressed in hematopoietic cells
    Mechanisms of Development, 2000
    Co-Authors: Michihiro Hidaka, William L Stanford, Georgina Caruana, Mehran Sam, Pamela H Correll, Alan Bernstein
    Abstract:

    Abstract Using an expression Gene Trapping strategy, we have identified and characterized two novel hematopoietic Genes, Hzf and Hhl . Embryonic stem (ES) cells containing a Gene trap vector insertion were cultured on OP9 stromal cells to induce hematopoietic differentiation and screened for lacZ reporter Gene expression. Two ES clones displaying lacZ expression within hematopoietic cells in vitro were used to Generate mice containing the Gene trap integrations. Paralleling this in vitro expression pattern, both Hzf and Hhl were expressed in a tissue-specific manner during hematopoietic development in vivo. Hzf encodes a novel protein containing three C 2 H 2 -type zinc fingers predominantly expressed in megakaryocytes and CFU-GEMM. Hhl encodes a novel protein containing a putative phosphotyrosine binding (PTB) domain expressed in megakaryocytes, CFU-GEMM and BFU-E. These results demonstrate the utility of expression Trapping to identify novel hematopoietic Genes. Future studies of Hzf and Hhl should provide valuable information on the role these Genes play during megakaryocytopoiesis.

  • expression Trapping identification of novel Genes expressed in hematopoietic and endothelial lineages by Gene Trapping in es cells
    Blood, 1998
    Co-Authors: William L Stanford, Michihiro Hidaka, Georgina Caruana, Katherine A Vallis, Maneesha S Inamdar, Victoria L Bautch, Alan Bernstein
    Abstract:

    We have developed a large-scale, expression-based Gene trap strategy to perform genome-wide functional analysis of the murine hematopoietic and vascular systems. Using two different Gene trap vectors, we have isolated embryonic stem (ES) cell clones containing lacZ reporter Gene insertions in Genes expressed in blood island and vascular cells, muscle, stromal cells, and unknown cell types. Of 79 clones demonstrating specific expression patterns, 49% and 16% were preferentially expressed in blood islands and/or the vasculature, respectively. The majority of ES clones that expressed lacZ in blood islands also expressed lacZ upon differentiation into hematopoietic cells on OP9 stromal layers. Importantly, the in vivo expression of the lacZ fusion products accurately recapitulated the observed in vitro expression patterns. Expression and sequence analysis of representative clones suggest that this approach will be useful for identifying and mutating novel Genes expressed in the developing hematopoietic and vascular systems.

  • aquarius a novel Gene isolated by Gene Trapping with an rna dependent rna polymerase motif
    Developmental Dynamics, 1998
    Co-Authors: Wolfgang Wurst, Mehran Sam, Alan Bernstein, Michael Kluppel, Ou Jin, Henry Heng
    Abstract:

    In a retinoic acid (RA) Gene- trap screen of mouse embryonic stem (ES) cells, a novel Gene, named Aquarius (Aqr), was identified and characterized. The promoterless lacZ marker was used to trap the genomic locus and to deter- mine the expression pattern of the Gene. Aqr transcripts are strongly induced in response to RA in vitro. During embryoGenesis, Aqr is ex- pressed in mesoderm, in the neural crest and its target tissues, and in neuroepithelium. Expres- sion was first detected at 8.5 days postcoitum, when neural crest cells are visible at the lateral ridges of the neural plate. The Gene-trapped Aqr locus was transmitted through the mouse germ line in three Genetic backgrounds. In the F2 Generation, the expected mendelian ratio of 1:2:1 was observed in all backgrounds, indicating that homozygous mice are viable. Homozygotes are normal in size and weight and breed normally. The Gene trap insertion, however, does not seem to Generate a null mutation, because Aqr tran- scripts are still present in the homozygous mu- tant animals. The Aqr open reading frame has weak homology to RNA-dependent RNA polymer- ases (RRPs) of the murine hepatitis viruses and contains an RRP motif. Aqr was mapped to mouse chromosome 2 between regions E5 through F2 by using fluorescence in situ hybridization analysis. Dev. Dyn. 1998;212:304-317. r 1998 Wiley-Liss, Inc.

Zongbin Cui - One of the best experts on this subject based on the ideXlab platform.

  • deficiency in the membrane protein tmbim3a grinaa initiates cold induced er stress and cell death by activating an intrinsic apoptotic pathway in zebrafish
    Journal of Biological Chemistry, 2019
    Co-Authors: Kai Chen, Guili Song, Yong Long, Tong Zhou, Shan Zhong, Zongbin Cui
    Abstract:

    Most members of the family of proteins containing a transmembrane BAX inhibitor motif (TMBIM) have anti-apoptotic activity, but their in vivo functions and intracellular mechanisms remain obscure. Here, we report that zebrafish Tmbim3a/Grinaa functions in the prevention of cold-induced endoplasmic reticulum (ER) stress and apoptosis. Using a Gene-Trapping approach, we obtained a mutant zebrafish line in which the expression of the tmbim3a/grinaa Gene is disrupted by a Tol2 transposon insertion. Homozygous tmbim3a/grinaa mutant larvae exhibited time-dependently increased mortality and apoptosis under cold exposure (at 16 °C). Mechanistically, using immunofluorescence, fluorescence-based assessments of intracellular/mitochondrial Ca2+ levels, mitochondrial membrane potential measurements, and Ca2+-ATPase assays, we found that cold exposure suppresses sarcoplasmic/ER Ca2+-ATPase (SERCA) activity and induces the unfolded protein response (UPR) and ER stress. We also found that the cold-induced ER stress is increased in homozygous tmbim3a/grinaa mutant embryos. The cold-stress hypersensitivity of the tmbim3a/grinaa mutants was tightly associated with disrupted intracellular Ca2+ homeostasis, followed by mitochondrial Ca2+ overload and cytochrome c release, leading to the activation of caspase 9- and caspase-3-mediated intrinsic apoptotic pathways. Treatment of zebrafish larvae with the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate-acetoxymethyl ester (BAPTA-AM) or with 2-aminoethoxydiphenyl borate (2-APB), an inhibitor of the calcium-releasing protein inositol 1,4,5-trisphosphate receptor (IP3R), alleviated cold-induced cell death. Together, these findings unveil a key role of Tmbim3a/Grinaa in relieving cold-induced ER stress and in protecting cells against caspase 9- and caspase 3-mediated apoptosis during zebrafish development.

  • novel strategies for Gene Trapping and insertional mutaGenesis mediated by sleeping beauty transposon
    Mobile genetic elements, 2013
    Co-Authors: Guili Song, Zongbin Cui
    Abstract:

    Gene and poly(A) Trappings are high-throughput approaches to capture and interrupt the expression of endogenous Genes within a target genome. Although a number of Trapping vectors have been developed for investigation of Gene functions in cells and vertebrate models, there is still room for the improvement of their efficiency and sensitivity. Recently, two novel Trapping vectors mediated by Sleeping Beauty (SB) transposon have been Generated by the combination of three functional cassettes that are required for finding endogenous Genes, disrupting the expression of trapped Genes, and inducing the excision of integrated traps from their original insertion sites and then inserting into another Gene. In addition, several other strategies are utilized to improve the activities of two Trapping vectors. First, activities of all components were examined in vitro before the Generation of two vectors. Second, the inducible promoter from the tilapia Hsp70 Gene was used to drive the expression of SB Gene, which can mediate the excision of integrated transposons upon induction at 37 °C. Third, the Cre/LoxP system was introduced to delete the SB expression cassette for stabilization of Gene interruption and bio-safety. Fourth, three stop codons in different reading frames were introduced downstream of a strong splice acceptor (SA) in the Gene Trapping vector to effectively terminate the translation of trapped endogenous Genes. Fifth, the strong splicing donor (SD) and AU-rich RNA-destabilizing element exhibited no obvious insertion bias and markedly reduced SD read-through events, and the combination of an enhanced SA, a poly(A) signal and a transcript terminator in the poly(A) Trapping vector efficiently disrupted the transcription of trapped Genes. Thus, these two Trapping vectors are alternative and effective tools for large-scale identification and disruption of endogenous Genes in vertebrate cells and animals.

  • effective expression independent Gene Trapping and mutaGenesis mediated by sleeping beauty transposon
    Journal of Genetics and Genomics, 2012
    Co-Authors: Guili Song, Yong Long, Perry B Hackett, Zongbin Cui
    Abstract:

    Expression-independent Gene or polyadenylation [poly(A)] Trapping is a powerful tool for genome-wide mutaGenesis regardless of whether a targeted Gene is expressed. Although a number of poly(A)-trap vectors have been developed for the capture and mutation of Genes across a vertebrate genome, further efforts are needed to avoid the 3'-terminal insertion bias and the splice donor (SD) read-through, and to improve the mutagenicity. Here, we present a Sleeping Beauty (SB) transposon-based vector that can overcome these limitations through the inclusion of three functional cassettes required for Gene-finding, Gene-breaking and large-scale mutaGenesis, respectively. The functional cassette contained a reporter/selective marker Gene driven by a constitutive promoter in front of a strong SD signal and an AU-rich RNA-destabilizing element (ARE), which greatly reduced the SD read-through events, except that the internal ribosomal entry site (IRES) element was introduced in front of the SD signal to overcome the phenomenon of 3'-bias Gene Trapping. The breaking cassette consisting of an enhanced splicing acceptor (SA), a poly(A) signal coupled with a transcriptional terminator (TT) effectively disrupted the transcription of trapped Genes. Moreover, the Hsp70 promoter from tilapia genome was employed to drive the inducible expression of SB11, which allows the conditional remobilization of a trap insert from a non-coding region. The combination of three cassettes led to effective capture and disruption of endogenous Genes in HeLa cells. In addition, the Cre/LoxP system was introduced to delete the Hsp70-SB11 cassette for stabilization of trapped Gene interruption and biosafety. Thus, this poly(A)-trap vector is an alternative and effective tool for identification and mutation of endogenous Genes in cells and animals.

  • effective Gene Trapping mediated by sleeping beauty transposon
    PLOS ONE, 2012
    Co-Authors: Guili Song, Yong Long, Perry B Hackett, Zongbin Cui
    Abstract:

    Gene Trapping is a high-throughput approach to elucidate Gene functions by disrupting and recapitulating expression of Genes in a target genome. A number of transposon-based Gene-Trapping systems are developed for mutaGenesis in cells and model organisms, but there is still much room for the improvement of their efficiency in Gene disruption and mutation. Herein, a Gene-Trapping system mediated by Sleeping Beauty (SB) transposon was developed by inclusion of three functional cassettes. The mutation cassette can abrogate the splice of trapped Genes and terminate their translation. Once an endogenous Gene is captured, the finding cassette independently drives the translation of reporter Gene in HeLa cells and zebrafish embryos. The efficiency cassette controls the remobilization of integrated traps through inducible expression of SB Gene. Analysis of transposon-genome junctions indicate that most of trap cassettes are integrated into an intron without an obvious 39 bias. The transcription of trapped Genes was abrogated by alternative splicing of the mutation cassette. In addition, integrated transposons can be induced to excise from their original insertion sites. Furthermore, the Cre/LoxP system was introduced to delete the efficiency cassette for stabilization of Gene interruption and bio-safety. Thus, this Gene-trap vector is an alternative and effective tool for the capture and disruption of endogenous Genes in vitro and in vivo.