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ES cell-derived mice are tools for rapid assessment of gene function

ES cell-derived mice are tools for rapid assessment of gene function. Generation of ES cell-derived embryos using tetraploid complementation technique. – Trophoblast – Endoderm of yolk sac. – Embryo proper – Mesoderm of yolk sac. EGFP tetraploid embryo. ES cells. (Courtesy of Andras Nagy).

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ES cell-derived mice are tools for rapid assessment of gene function

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  1. ES cell-derived mice are tools for rapid assessment of gene function

  2. Generation of ES cell-derived embryosusing tetraploid complementation technique – Trophoblast – Endoderm of yolk sac – Embryo proper – Mesoderm of yolk sac EGFP tetraploid embryo ES cells (Courtesy of Andras Nagy)

  3. Hybrid ES cells make mice Andras Nagy and Marina Gertsenstein

  4. ES-derived mice allow phenotypic analysis without germ line transmission • Dominant transgenes • Gain of function targeted mutations • Affinity tagging target genes • siRNA knock-down approaches

  5. Timelines to phenotype Vector production 4weeks 1 week Electroporation and 8weeks 5 weeks clone selection Live chimera production 11weeks 8 weeks Germline transmission 22 weeks na Heterozygous crosses 30 weeks na Phenotype 33 weeks8 weeks Gene targeting siRNA

  6. Affinity-tagging of target genes • Targeted insertion of 3’ double affinity tag • Transcription factors • Identification of protein interactions and DNA binding sites • Disease-related genes • Protein interaction partners in disease-related tissues • Novel targets for therapeutics

  7. KOMP- a mutation resource for the mouse genome • Gene trap with a variety of vectors until return on investment too poor • Undertake complementary high-throughput gene targeting • Maintain and distribute ES cell resource • Complementary to Regulome- generate tagged alleles

  8. NorCOMM: High Throughput Mammalian Functional Analysis for the Discover of Novel Determinants of Human Disease Geoff Hicks and Janet Rossant

  9. Key Scientific Objectives and Milestones • Knockout Mouse Resource • NorCOMM: 144,000 gene trap lines. Complemented with 500 targeted lines. (Hicks, Stanford, Lefebvre) • EuCOMM 120,000 gene trap lines. Complemented with 8000 targeted lines. • Genetic ToolboxNovel technologies to enhance use of the Knockout Mouse Resource. (Nagy, Stanford, Ishida) • Repository and Distribution CentreRobust resource to archive and distribute all components to the broad scientific community (McKerlie)

  10. Number of NCBI sequence submissions by IGTC gene trapping centres

  11. pb-geo En-2 ---- pA LacZ pA SA - Most widely used type of vector, all neo resistant clones are in genes - Only genes expressed in ES cells can be targeted pA SA b-geo pb-gal/PT1 En-2 ---- • first vector used to target all genes regardless of expression • - inefficient since many neo insertions not in genes En-2 pax2 pMS1 Stop Stop Lox P Lox P PGK PGK PGK Bsd Neo Neo pA pGTlox4 SD En-2 HPRT SA LacZ • polyA trap vectors- target all genes regardless of expression • clone gene by 3’RACE • query on mutagenicity IRES pA SA LacZ SD VectorCartoon SA SD

  12. UPATrap overcomes 3’ insertional bias by overcoming nonsense-mediated decay of the selectable marker fusion transcript Cre NMD Shigeoka et al., (2005) Nucleic Acids Res. 33:20

  13. NMDi retains the 3’ insertional bias and utilizes nonsense-mediated decay for mutagenesis NMD Cre Epp and Stanford

  14. NMDf vectors also allow reversion of the mutated locus to a protein-tagged wild-type allele NMD Cre

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