By homologous recombination in ES cells heterozygous for the Nodaltm1Rob allele, the entire 600 bp ASE enhancer region was replaced by a loxP-flanked hygromycin selection cassette in trans to the original mutation. Transient expression of Cre recombinase in targeted ES cells resulted in excision of the selection cassette, leaving a single loxP site in place of the enhancer sequences. Subsequent matings of mice carrying this allele resulted in segregation from the Nodaltm1Rob allele.