Using homologous recombination in a bacterial artificial chromosome (BAC, clone RPCI23-331K23), an FRT-flanked neo cassette (followed by a loxP site) was inserted immediately downstream of exon 8 of the targeted gene. An additional loxP site was added upstream of exon 8 when the targeting vector was excised from the BAC. Correctly targeted ES cells were injected into blastocysts and chimeric mice were bred to Flpe-expressing mice to excise the neo cassette.