A CMV-hyTK selection cassette flanked by two loxP sites was inserted approximately 2.6 kb upstream of exon 1. A third loxP site was inserted into intron 1. The hygromycin cassette was deleted in ES cells by transient transfection with a vector driving expression of Cre recombinase. The resulting ES cell derivatives with two loxP sites flanking exon 1 were selected and injected into blastocysts to generate the Vhltm1Jae allele.