D6177222d01 - 7.9. Cell ablation via cre-loxP - Logical...

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7.9. Cell ablation via cre-loxP - Logical application - e.g. DTA gene with floxed STOP (or LacZ) and artificial - intron in ROSA26 locus - Then cross with tissue specific cre
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7.10. Flp - FRT - alternative site-specific recombination system - from yeast - flp is recombinase - FRT sites resemble loxP: core 8 bp, 2 IR of 13 bp - recombinase is a tetramer -t opt is 20°C: very un-efficient in ES cells - But now mutant flp: flp-e: t opt 37°C
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Flp activity reporter mouse Flp-FRT detection flp expression pattern: see cre-ROSA26 system: ROSA26 locus + FRT/PGK-NeoR-PLAP Works fine
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Combination Flp-cre Logical way to make floxed alleles, e.g. for flox-and-delete
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Combination Flp-cre: RMCE To generate several mutants of a certain exon in ES cells First: target exon with PSM-NSM floxed with heterotypic LoxP’s Then: via site-specifieke R between LoxP’s new exons only LoxP’s as homology!! TV also PSM: flipped-NeoR VERY efficient
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4 Hyg-HSV-tk loxP loxP511 FRT-neo-FRT * FRT-neo-FRT * cre flp HR-TV = RMCE
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7.11. Problems with cre recombinase Discussion: cryptic loxP-like sequences in genome?! In vivo: protamine-1 -promotor-cre TG mouse Strong expression in spermatids Male TG animals: sterile Male offspring of female TG mice: also sterile All female TG mice: fertile LoxP-independent recombination with clear phenotype Probably at LoxP-like loci in genome during meiosis
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7.12. Place specific knock-in of BAC clones Advantage BAC in TG: see earlier But: random integration…
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D6177222d01 - 7.9. Cell ablation via cre-loxP - Logical...

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