Generation and characterization of pCPB-deficient mice. (a) Design of the gene-targeting vector. Upon homologous recombination, a mutated pCPB allele was created that contained the neo resistance gene inserted in exon 2. (b) Southern blot analysis of pCPB gene disruption in ES cells. The 80-mer probe (3 in a) detected an approximately 9-kb fragment from the wild-type allele, whereas the disrupted allele produced a 4.3-kb fragment after ApaI digestion of genomic DNA. (c) PCR analysis of mouse tail tip DNA. Mice carrying a mutant pCPB gene were identified using a neo-specific primer (2a in a) and a primer located with exon 3 (2b in a). The PCR amplification produced a product of about 3 kb in pCPB+/– and pCPB–/– mice. Homozygote animals were identified by an additional PCR analysis. An exon primer pair situated 5′ (1a in a) and 3′ (1b in a) of the neo insertion site amplified a 120-bp fragment in pCPB+/+ and pCPB+/– animals only. (d) Northern blot analysis of pCPB transgenic mice. Total RNA was isolated from 6-week-old mouse livers of pCPB+/+, pCPB+/–, and pCPB–/– animals. Hybridization with a 600-bp random-labeled pCPB DNA fragment revealed a 1.4-kb band present in pCPB+/+ and pCPB+/– animals, but absent from pCPB–/– animals.