Targeted disruption of Fliih. (A) The structures of the targeting vector, the relevant portion of the Fliih gene, and the targeted allele after homologous recombination are depicted. Restriction enzyme sites are indicated (B, BspEI; E, EcoRV; K, KpnI; N, NcoI; Ps, PshAI; Pv, PvuI; and X, XhoI). The asterisk denotes the BspEI site in exon 5 introduced by site-directed mutagenesis. Fliih exons are depicted by the numbered open boxes. The tk-neo and pgk-thymidine kinase cassettes and the pBluescript vector are indicated. The dotted lines indicate the regions of identity between the targeting vector and the wild-type Fliih allele. (B) Southern analysis of targeted ES cell lines. Genomic DNA was digested with NcoI and EcoRV, electrophoresed, blotted to a nitrocellulose membrane, and hybridized to the [32P]-labeled probe fragment indicated in panel A. Untargeted ES cell DNA was run as a control (BALB/c). The three lines injected into blastocysts to generate chimeric mice are indicated by asterisks. Sizes of bands (in kilobases) are indicated. (C) PCR results on individual blastocysts. Sizes of bands (in kilobases) are indicated.