Targeting took place in ES cells carrying a cre transgene Tg(Prm-cre)70Og, which expresses Cre recombinase under control of the male-germline specific protamine promoter. A floxed neo cassette replaced the coding region for the entire homeodomain and CVC domain, leaving fragments of exons 1 and 5 intact. Cre-mediated excision removed the neo cassette upon transmission from male chimeras.