Strategy for targeting the mouse factor IX gene by homologous recombination. (Top) Endogenous gene locus. Exons 1 to 5 of the endogenous mouse factor IX gene are shown. (Middle) Targeting construct. The construct linearized with PmeI and used for homologous recombination is shown. Recombination between the 5′ (1.5 kb) and 3′ (5.3 kb) regions of homology is depicted. The PmeI site is within the plasmid vector, 29 nucleotides from the BamHI site. The human factor IX cDNA is depicted in black. (Bottom) Targeted gene locus. The mouse factor IX locus after homologous recombination is shown. The regions from which the 730-bp and 680-bp probes were generated are shown. After EcoRV cleavage of mouse gDNA, the 730-bp probe gives a 4.7-kb fragment and the 680-bp probe a 13.4-kb fragment when homologous recombination has occurred. Mouse gDNA is shown as a thick line and the inserted neo sequences as a thinner line. B indicates BamHI; P, PacI.