A targeting cassette containing IRES-cre/ERT2-pA and FRT-flanked PGK-neo-bpA sequences was introduced into AB1 ES cells. Homologous recombination inserted into the 3' UTR of exon 3 of the Sox9 locus. Targeted ES cells were injected into C57BL/6 blastocysts to produce chimeras which were crossed with C57BL/6 wild-type animals. The neo cassette was not removed from the targeted allele.