Scheme for the homologous recombination between the targeting vector (socket/F9) and the endogenous factor IX gene of the murine ES cells. Homologous recombination causes replacement of a 5.5-kb segment that includes the promoter to exon 3 region of the factor IX gene with the socket (neoΔHPRT) in the ES cells. Restriction sites changed by the homologous recombination caused the targeted DNA to yield a 7-kb instead of the wild-type 14-kb fragment after BamHI digestion and a 12-kb instead of the wild-type 8.8-kb fragment after Bgl II digestion. The G418 resistance gene (neo ) and the thymidine kinase gene (TK ) are used for positive and negative selection, respectively. Locations of PCR primers and the PE449 probe for Southern blot analysis are indicated. Thick black lines indicate genomic DNA and regions of homology. Exons 1, 2, 3, and 4 of the murine factor IX gene are labeled. B, BamHI; B′, Bgl II.