A loxP site was inserted in intron 2 and a floxed CMV-Hygro selection cassette was inserted in intron 3. ES cells were sequentially targeted so that ES cells carried two mutated alleles. Cre recombinase treatment of these ES cells resulted in two mutant alleles, this one with a floxed exon 3 but lacking the selection cassette and another allele which lacks both exon 3 and the selection cassette.