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Fig. 3. The generation of Corin knockout mice. (A) Gene targeting approach used for Corin ablation. A YFP-Neo cassette replaced 64 bp downstream of the ATG in exon 1 (red box). Primers (arrowheads, see Table 1) and expected PCR fragments are indicated. Primer WT51 binds in the deleted 64 bp segment and the primers SC3 and SC5 bind outside the targeting construct. The NcoI fragments used for Southern analysis are indicated. The FRT flanked pgkNeo^R cassette was oriented in parallel to Corin transcription. (B,C,D) Genomic analysis of wild-type, heterozygous and homozygous Corin mice. (B) Southern analysis of NcoI-digested genomic DNA. The 5'-probe reveals 18.6 kb and 10 kb bands from wild-type and mutant alleles, respectively. (C) PCR with primers SCNEO5 and SC3 generates a 6075 bp band from the targeted allele (upper panel) whereas WT51 and SC3 generate a 5266 bp fragment from the wild type. (D) PCR with WT51, WT31, KILY5 and SEQFP1 detects both wild-type (312 bp) and mutant (478 bp) alleles for routine genotyping. (E,F) Immunostaining of frozen P3 skin-sections from wild-type and mutant mice with anti-Corin antibodies. Corin, green; Nuclei, red.

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