Methodology

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

Thomas M Wishart^1,2 , Stephen HF MacDonald³ , Philip E Chen² , Michael J Shipston¹ , Michael P Coleman⁴ , Thomas H Gillingwater^1,2 and Richard R Ribchester²

¹Centre for Integrative Physiology, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh, UK

²Centre for Neuroscience Research, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh, UK

³Trinity Institute of Molecular Medicine, St. James's Hospital, Dublin 8, Ireland

⁴Laboratory of Molecular Signalling, Babraham Institute, Babraham, Cambridge, UK

author email corresponding author email

Molecular Neurodegeneration 2007, 2:21doi:10.1186/1750-1326-2-21

Published:	30 October 2007

Abstract

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.