A targeting vector was constructed where an upstream loxP site was cloned into a blunted NsiI site in intron 7 and a floxed neomycin resistance cassette was cloned into a BglII site in intron 9. Targeting took place in PC3 ES cells homozygous for the cre transgene Tg(Prm-cre)70Og, which expresses Cre recombinase in male germ cells. Cre mediated recombination subsequently removed only the neomycin cassette upon transmission from male chimeric mice. Offspring from the male chimeras in which exons 8 and 9 remained floxed were selected for further breeding. Expression was unaffected as determined by immunoblot analysis of splenic extracts.