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

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

Hybrid ES cells make mice Andras Nagy and Marina Gertsenstein

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

Timelines to phenotype Vector production 4weeks1 week Electroporation and 8weeks5 weeks clone selection Live chimera production 11weeks8 weeks Germline transmission 22 weeksna Heterozygous crosses 30 weeksna Phenotype 33 weeks 8 weeks Gene targetingsiRNA

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

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

Competition III Science Review NorCOMM: High Throughput Mammalian Functional Analysis for the Discover of Novel Determinants of Human Disease Geoff Hicks and Janet Rossant

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 Toolbox Novel technologies to enhance use of the Knockout Mouse Resource. (Nagy, Stanford, Ishida) Repository and Distribution Centre Robust resource to archive and distribute all components to the broad scientific community (McKerlie)

Number of NCBI sequence submissions by IGTC gene trapping centres

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

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

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

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