A 0.5 kb region of the gene containing the asymmetric enhancer (ASE) was replaced with a loxP site flanked neomycin resistance gene cassette via homologous recombination. The floxed neo cassette was subsequently removed by crossing chimeric animals to a cre-expressing line. Ablation of asymmetric gene expression in homozygous mutant embryos was confirmed by whole mount in situ hybridization.