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

Generation of ES cell-derived embryosusing 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 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 weeks 8 weeks

Gene targeting siRNA

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 IIIScience 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 pax2SA LacZ PGK SDNeopAIRES

SA -geo pAp-geo En-2 ----

- Most widely used type of vector, all neo resistant clones are in genes- Most widely used type of vector, all neo resistant clones are in genes- Only genes expressed in ES cells can be targeted- Only genes expressed in ES cells can be targeted

SA LacZ pA PGK Neo pAp-gal/PT1 En-2 ----

- first vector used to target all genes regardless of expressionfirst vector used to target all genes regardless of expression- inefficient since many neo insertions not in genes- inefficient since many neo insertions not in genes

pGTlox4

Stop

SA LacZ PGK SDBsd

Stop

pALox PLox P

En-2 HPRT

- polyA trap vectors- target all genes regardless of expressionpolyA trap vectors- target all genes regardless of expression- clone gene by 3’RACEclone gene by 3’RACE- query on mutagenicityquery 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