The gene was disrupted by replacement of the 5' portion of exon 5 with a PGK-neo cassette via homologous recombination. RT-PCR analysis using primers to exons 3-4 and exons 3-5 did not detect gene expression in the midbrain and diencephalons of homozygous mutant animals. In situ hybridization of parasagittal brain sections from homozygous mutants using a probe to the deleted portion of the gene did not detect gene expression. The null allele was further demonstrated by radioimmunoassay (RIA) of brain samples from homozygous mutant animals, which did not detect protein product.