FIG. 3.
Targeted disruption of the mouse C2β gene. (A) Targeting strategy. Partial restriction enzyme maps and schematic representation of the strategy for ablation of the C2β gene. The PIK3C2B gene of 129Sv mice (wild-type [WT] allele), the targeting vector, the recombinant loxP-flanked locus (floxed allele), and the C2β exon-deleted locus (deleted allele) are shown. Cleavage sites for BamHI (B) and HindIII (H) are marked. The locations of probes 1 and 2 for Southern analysis are indicated by closed bars, and primers for PCR analysis are shown as arrowheads. Exons are symbolized as numbered rectangles. Exon 1 encodes an untranslated region; the start codon is located in exon 2. (B) Southern blot analysis of BamHI-digested genomic DNA and hybridization to probe 1 reveal a 6-kb and an 8-kb band corresponding to wild-type and floxed alleles, respectively. (C) Southern hybridization for detection of the null allele. The HindIII fragments in the wild-type allele and deleted alleles are 8.3 kb and 5.8 kb, respectively, when detected with probe 2. (D) Mice were genotyped by PCR. The amplicon with primer pair 1 and 2 is 251 bp for the wild-type allele. The amplicon with primer pair 3 and 4 is 404 bp for the null allele. (E) Loss of normal C2β mRNA transcript in C2β−/− mice. RNA was isolated from cultured keratinocytes. The forward primer was designed in exon 2, and the reverse primer was designed in exon 7. The wild-type allele yields a 678-bp amplicon, and the deleted allele yields a 301-bp amplicon. (F) Loss of epidermal C2β protein in C2β−/− mice. Western blot analysis showing absence of C2β protein expression in the knockout mice. Proteins were isolated from epidermal extracts and detected with an antiserum to C2β. (G) Loss of C2β protein in visceral tissues of C2β−/− mice.