Generation of the

FvR504Q “knock-in” allele by gene targeting.(A) Structure of the FV gene (from exons 7 through 13), targeting vector carrying the R504Q mutation in exon 10 and selectableTK/neo cassette flanked by loxP sites, and the expected results of successful homologous recombination and Cre excision of the TK/neo cassette. (B) Southern blot analysis—using the exon 13 probe indicated in panel A—of DNA prepared from targeted ES cells following digestion with EcoRV. Lanes 1 and 2 show only the germline band at 23 kb. Lane 3 shows a successfully targeted ES clone with the predicted 12.5-kb band from the recombined allele and the 23-kb band from the remaining allele. (C) Southern blot analysis using same probe as in panel 1B to detect ES clones that had undergone Cre-mediated excision as shown in lanes 3 and 5 by the absence of the 12.5-kb EcoRV fragment. (D) PCR analysis of wild-type and mutant Fv alleles using primers that cross the insertion site in intron 10. Amplification of DNA from the wild-type allele yields a 124-bp fragment, and DNA from the mutant allele produces a 263-bp fragment.