A loxP site was introduced to intron 3 and an FRT-PGK-Neo-pA-splice donor-FRT-loxP cassette was placed in intron 4. Flpe-mediated recombination removed the FRT-flanked neo cassette leaving loxP sites on either side of exon 4. Cre-mediated recombination removed exon 4 and introduced a premature stop codon. Western blot analysis demonstrated that no detectable protein was present in homozygous E13.5 embryos.