By homologous recombination in ES cells heterozygous for the Nodaltm1Rob allele, 100 bp of the ASE enhancer region (including the two 10 bp binding sites) was replaced by a loxP-flanked hygromycin selection cassette in cis 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.