Figure 1.

Generation of conditional caspase 8 mutant mice. (A) Schematic representation of the wild-type caspase 8 locus, the targeting construct, and the caspase 8 mutant alleles casp8^fl3-4;neo-tk, casp8^fl3-4, and casp8^Δ3-4. Exons are indicated as filled boxes and LoxP sites as triangles. B, BamHI site. (B) Four independent clones carrying homologous recombination to the caspase 8 locus were identified by PCR and confirmed by Southern blot with the 5′ probe. (Lane 1) Wild-type ES DNA. (Lanes 2–5) casp8^fl3-4;neo-tk ES clones. (C) casp8^fl3-4,neo-tk ES clones were transiently transfected with a CMV-driven construct encoding for Cre recombinase. Clones that have lost the neo-tk cassettes but retained the floxed caspase 8 exons 3 and 4 (casp8^fl3-4) were identified by Southern blot with exons 3–4.(Lane 1) Wild-type ES clone. (Lane 2) casp8^{fl3-4;neo-tk/wt} ES clone. (Lane 3) casp8^fl3-4/wt ES clone. (Lane 4) casp8^Δ3-4/wt ES clone. (D) Heterozygous (casp8^fl3-4/wt) mutant littermate mice were identified by Southern blot on tail DNA with a 5′-flanking probe. (Lanes 1,3,4) casp8^fl3-4/wt. (Lane 2) Wild type. BamHI restriction fragments (detected by 5′ probe): wild-type fragment = 9.4 kb; casp8^fl3-4;neo-tk = 8.7 kb; casp8^fl3-4 = 4.5 kb; and casp8^Δ3-4 = 9.0 kb.