Design of a novel quantitative PCR (QPCR)-based protocol for genotyping mice carrying the neuroprotective Wallerian degeneration slow (Wld(s)) gene

BACKGROUND: Mice carrying the spontaneous genetic mutation known as Wallerian degeneration slow (Wld(s)) have a unique neuroprotective phenotype, where axonal and synaptic compartments of neurons are protected from degeneration following a wide variety of physical, toxic and inherited disease-inducing stimuli. This remarkable phenotype has been shown to delay onset and progression in several mouse models of neurodegenerative disease, suggesting that Wld(s)-mediated neuroprotection may assist in the identification of novel therapeutic targets. As a result, cross-breeding of Wld(s )mice with mouse models of neurodegenerative diseases is used increasingly to understand the roles of axon and synapse degeneration in disease. However, the phenotype shows strong gene-dose dependence so it is important to distinguish offspring that are homozygous or heterozygous for the mutation. Since the Wld(s )mutation comprises a triplication of a region already present in the mouse genome, the most stringent way to quantify the number of mutant Wld(s )alleles is using copy number. Current approaches to genotype Wld(s )mice are based on either Southern blots or pulsed field gel electrophoresis, neither of which are as rapid or efficient as quantitative PCR (QPCR). RESULTS: We have developed a rapid, robust and efficient genotyping method for Wld(s )using QPCR. This approach differentiates, based on copy number, homozygous and heterozygous Wld(s )mice from wild-type mice and each other. We show that this approach can be used to genotype mice carrying the spontaneous Wld(s )mutation as well as animals expressing the Wld(s )transgene. CONCLUSION: We have developed a QPCR genotyping method that permits rapid and effective genotyping of Wld(s )copy number. This technique will be of particular benefit in studies where Wld(s )mice are cross-bred with other mouse models of neurodegenerative disease in order to understand the neuroprotective processes conferred by the Wld(s )mutation.