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

Abstract

<p>Abstract</p> <p>Background</p> <p>Mice carrying the spontaneous genetic mutation known as Wallerian degeneration slow (<it>Wld</it>^<it>s</it>) 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 <it>Wld</it>^<it>s</it>-mediated neuroprotection may assist in the identification of novel therapeutic targets. As a result, cross-breeding of <it>Wld</it>^{<it>s </it>}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 <it>Wld</it>^{<it>s </it>}mutation comprises a triplication of a region already present in the mouse genome, the most stringent way to quantify the number of mutant <it>Wld</it>^{<it>s </it>}alleles is using copy number. Current approaches to genotype <it>Wld</it>^{<it>s </it>}mice are based on either Southern blots or pulsed field gel electrophoresis, neither of which are as rapid or efficient as quantitative PCR (QPCR).</p> <p>Results</p> <p>We have developed a rapid, robust and efficient genotyping method for <it>Wld</it>^{<it>s </it>}using QPCR. This approach differentiates, based on copy number, homozygous and heterozygous <it>Wld</it>^{<it>s </it>}mice from wild-type mice and each other. We show that this approach can be used to genotype mice carrying the spontaneous <it>Wld</it>^{<it>s </it>}mutation as well as animals expressing the <it>Wld</it>^{<it>s </it>}transgene.</p> <p>Conclusion</p> <p>We have developed a QPCR genotyping method that permits rapid and effective genotyping of <it>Wld</it>^{<it>s </it>}copy number. This technique will be of particular benefit in studies where <it>Wld</it>^{<it>s </it>}mice are cross-bred with other mouse models of neurodegenerative disease in order to understand the neuroprotective processes conferred by the <it>Wld</it>^{<it>s </it>}mutation.</p