Insertional mutation of the CBP gene. (A) The structures of the trapping vector (top), the wild-type allele (middle), and the mutant allele (bottom) are shown. The trapping vector, pU-San, contains two loxP sequences, a splice acceptor region (SA) of the mouse En-2 gene, an internal ribosomal entry site (IRES), a β-galactosidase/neomycin phosphotransferase fusion gene, the SV40 polyadenylation sequence (β-geo-pA), and the pUC19 vector as indicated. The gray boxes represent exons. Restriction enzyme sites (B, BamHI; E, EcoRI; H,HindIII; S, SphI), the location of probes (bars) used to confirm single integration of trapping vector, and the expected fragment sizes are indicated. Probe A is anSpeI-BgIII fragment in the SA of the trapping vector; probe B is an ScaI-XbaI fragment in pUC sequences of the trapping vector; probe C is theHindIII-XhoI fragment immediately upstream of the vector integrated region. The open and closed arrowheads indicate the location of primers used in RT-PCR for genotyping. (B) Southern blot analysis of an Ayu-San112 ES clone and a normal TT2 ES cell. (Left) A blot using HindIII (H) or SphI-digested (S) DNA hybridized with probe A. (Middle) A blot BamHI- (B) or EcoRI-digested (E) DNA with probe B. (Right) A blot of using EcoRI-restricted DNA hybridized with probe C. Molecular-weight makers are shown on the right. The bars indicate the positions of size marker: 23130, 9416, 6557, 4361, and 2322 bp from the top.