A loxP site was inserted upstream of exon 2 and a loxP site and FRT flanked neo cassette were inserted downstream of exon 2 via homologous recombination. Flp mediated recombination removed the neo cassette. Cre mediated recombination deleted exon 2. Semi-quantitative PCR and Western blot analysis of whole-cell extracts and conditioned medium confirmed the absence of transcript and protein expression in MEFs derived from homozygous mutant mice.