A PGK-neo cassette was inserted to replace the first 750 nucleotides of the 5' region, the initiation codon, and the first 17 codons of the basic helix-loop-helix (bHLH) domain. While message was present in homozygous mutant mice at levels only slightly below normal, immunocytochemistry analysis showed an absence of protein product in the coronal sections of the brain.