A gene trap cassette, consisting of an FRT site, an engrailed 2 splice acceptor site, IRES, EGFP, a loxP site, a neomycin selection gene, another FRT site and a loxP flanked exon 5, was inserted into intron 4. Flp-mediated recombination removed the EGFP and neomycin gene, leaving exon 5 floxed. Exon 5 was removed via cre-mediated recombination in the germline resulting in a frameshift and premature stop codon in exon 6. qPCR confirmed the absence of mRNA in homozygotes.