A cre/ERT2-polyA sequence was inserted at the translation start site of the Nkx3-1 gene, thus generating a null allele. The targeting vector also contained a self-excising ACE-cre/PolII-neo selection cassette for positive selection and a PGK-tk (thymidine kinase) cassette for negative selection. The vector was transfected into TC-1 ES cells which were used to generate chimeras. Excision of the selection cassette occurred by passage through the male germline.