Development of an efficient method for genotyping at the gd locus in NC/Sgn mice based on PCR-RFLP analysis

Growth deficit (gd) is a recessive mutation that occurs spontaneously in the inbred NC/Sgn mouse strain. Because homozygotes (gd/gd) of both sexes are sterile, they must be produced by mating putative heterozygous carriers (+/gd) whose phenotypes are essentially the same as those of wild-type +/+ mice. The objectives of this study were to develop an efficient method that distinguished a gd allele from a wild-type allele and, if possible, to identify nucleotide substitutions responsible for the gd mutation. The location of the gd locus was estimated to be at 58.3 Mbp on chromosome 4, over which Musk is located. An A-to-G base substitution, which resulted in an M826V amino acid exchange, was identified within a tyrosine kinase domain of Musk. This base substitution disrupted a recognition site for NlaIII; this allowed for discriminating the gd allele from the wild-type allele using PCR-RFLP analysis. When 130 (C57BL/6J × NC/Sgn-gd) F(2) mice were genotyped by PCR-RFLP analysis, all 32 growth-retarded F(2) mice were judged to have the gd/gd genotype. Musk mutations are known to cause congenital myasthenia, which is accompanied by growth retardation, postnatal lethality, and development of a hunchback. These were the typical phenotypes of gd/gd mutants. Although we cannot rule out the possibility that the neighboring genes around the Musk locus are related to the gd phenotype, gd could possibly be classified as a mutant allele of Musk.