100 bp of exon 2, encoding most of the first transmembrane domain, was replaced by an IRES-geo-neo cassette. The insertion was 54 bp after the start codon of the endogenous coding sequence, and the presence of stop codons in three reading frames upstream of the IRES-geo-neo allowed interruption of translation of the endogenous mRNA 18 amino acids after the beginning. Southern blot confirmed recombination and Western blot indicated lack of wild-type protein in mutant brains.