sEPCR gene targeting. (A) A schematic representation of the targeted Procr gene mutation (sEPCR: Leu215Stop) using the Cre/Lox system. Neomycin gene floxed by 2 loxP sites and a BclI restriction site (sEPCR: Leu215Stop) were incorporated in the Procr loci after homologous targeting. Subsequently, the neo^R gene was deleted following transient transfection with a Cre expression vector. (B) Southern blot of ES clone DNA. After homologous targeting, the DNA was digested with BclI. The sEPCR mutant allele gave a 5.1-kb band, whereas the wild-type allele gave a 12.5-kb band with probe “a.” When the probe “b” was used, the mutant allele gave 6.9-kb and 2.2-kb bands due to a BclI site in the Neo gene and the integration of another BclI site in exon 4. After Cre transfection, the DNA was digested with BclI. The Procr^s/+ clones showed 12.5-kb and 5.7-kb bands when hybridized with probe “a.” When the probe “b” was used, the mutant allele gave 6.9-kb and 5.7-kb bands, due to the integration of a BclI site in exon 4 of the Procr gene. (C) PCR genotyping. After PCR amplification, PCR products (1000 bp) were digested with BclI. For the Procr^s/+ mutation, 1000-bp/300-bp and 700-bp bands were visualized. For Procr^s/s embryos, only 700-bp and 300-bp bands were visualized.