A loxP-flanked neomycin resistance cassette with TK promoter was introduced into intron 3. Transient Cre expression removed the Neo cassette, leaving a residual intron 3 loxP site. The ES cells were re-targeted to introduce a loxP-flanked neomycin cassette into intron 1. Transient Cre expression excised this neo cassette, leaving exons 2 and 3 flanked by loxP sites. (J:101977)