EUCOMM • Major objective: • Provision of floxed alleles for all coding genes • Establishment of mice • Further development of Cre-driver strategies • Capitalize on ES cell resource – cellular screens – homozygous ES cells The Challenge • 20,000 floxed alleles • Inducible Cre driver strains? EUCOMM Cre-driver effort: Transgene Type of Transgene Construct Status Expression Genetic Background ICS Ins-CreERT2 Promoter done Pancreatic b cells C57BL/6J (96%) ICS Tph2-CreERT2 BAC done Raphe serotonergic neurons C57BL/6J (95%) ICS Sox2-CreERT2 BAC done Neural Stem Cells ICS Lmx1a-CreERT2 BAC done Dopaminergic Neurons C57BL/6N (100%) ICS Pet1-CreERT2 BAC done Serotonergic Neurons C57BL/6N (100%) C57BL/6N (100%) Institute C57BL/6J (100%) ICS Sox9-CreERT2 BAC done Condensing mesenchyme and neural progenitors ICS Dll1-CreERT2 BAC done Delta 1 positive cells C57BL/6N (100%) EMBL Wnt8b-Cre Promoter done Medial dorsal cortical neurons C57BL/6J EMBL MLC-Cre Promoter done Fast muscle fibers C57BL/6J EMBL MLC-CreERT2 Promoter in progress Fast muscle fibers C57BL/6N EMBL bMyHC-CreERT2 Promoter In progress Slow muscles fibers C57BL/6N EMBL Pax7-CreERT2 Promoter in progress Satellite cells C57BL/6N C57BL/6N EMBL Plzf-CreERT2 BAC In progress Spermatogonial Stem Cell & Hematopoeitic progenitors) EMBL REC8-Cre BAC done Meiotic cells C57BL/6N EMBL TNP1-Cre BAC done Round Spermatids C57BL/6N RMCE with inducible CreERT2 Frt RMCE vector gene trap allele attB SA P2A CreERT2 attP IRES puro Frt F3 lx 5171 exon 1 F3 pA lx 5171 Frt F3 SA βgeo pA exon 2 Flp Frt knock-in allele exon 1 attB SA P2A CreERT2 attP IRES puro F3 pA exon 2 RMCE with inducible CreERT2 Frt RMCE vector attB SA P2A CreERT2 Frt IRES Frt pA F3 IRES puro F3 puro Cotransfection with a Flpe expression vector without IRES Puro frame 1 pA attP IRES frame 0 pA attP attB SA P2A CreERT2 Flpe puro attB SA P2A CreERT2 prom. attP F3 pA frame 2 RMCE with inducible CreERT2 Due to slow clone distribution Production of 150 rsFlipROSAbgeo* clones LacZ staining Selection of blue clones D7 F9 E11 RMCE with inducible CreER RMCE with RMCE Cre vector Frt attB SA P2A CreERT2 D7 attP IRES puro F9 F3 pA E11 RMCE with inducible CreERT2 RT-PCR on RNA from F9 Myst2 subclones Frt F3 lx 5171 gene trap allele exon 1 lx 5171 Frt F3 SA K55 exon 1 attB SA P2A CreERT2 K56 1 exon 2 B34 Frt RMCE allele pA βgeo attP IRES puro F3 pA exon 2 K38 2 3 4 5 7 8 9 10 11 12 13 14 RT-PCR Myst2-βgeo fusion (B34/K55 220 bp) RT-PCR Myst2-Cre fusion (K38/K56 377 bp) RMCE with inducible CreERT2 Activity of CreERT2: Cells transfected with loxP loxP PGK puro pA βgeo pA splitted onto two wells (identical cells) one well with 4OHT one well without 4OHT loxP loxP PGK puro pA loxP βgeo pA PGK white βgeo blue Frt attB SA P2A CreERT2 attP IRES puro F3 pA pA RMCE with inducible CreERT2 Activity of CreERT2: Clone F9 B12 -4OHT loxP PGK +4OHT βgeo pA Future: • Extent Cre-zoo: – Knock-ins, Bac- knock-ins, Enhancer identification, Transposon tagging • Technology development: – Intersection inactivation (A. Nagy) – Self-excision ((F.Stewart) • Inducible systems: – Alternatives to CreERT2 ? – rtTA, tTA systems ? – Lac operon (H. Scrabble)? – Others?
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