Abstract
BackgroundMice carrying the spontaneous genetic mutation known as Wallerian degeneration slow (Wlds) 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 Wlds-mediated neuroprotection may assist in the identification of novel therapeutic targets. As a result, cross-breeding of Wlds 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 Wlds mutation comprises a triplication of a region already present in the mouse genome, the most stringent way to quantify the number of mutant Wlds alleles is using copy number. Current approaches to genotype Wlds mice are based on either Southern blots or pulsed field gel electrophoresis, neither of which are as rapid or efficient as quantitative PCR (QPCR).ResultsWe have developed a rapid, robust and efficient genotyping method for Wlds using QPCR. This approach differentiates, based on copy number, homozygous and heterozygous Wlds mice from wild-type mice and each other. We show that this approach can be used to genotype mice carrying the spontaneous Wlds mutation as well as animals expressing the Wlds transgene.ConclusionWe have developed a QPCR genotyping method that permits rapid and effective genotyping of Wlds copy number. This technique will be of particular benefit in studies where Wlds mice are cross-bred with other mouse models of neurodegenerative disease in order to understand the neuroprotective processes conferred by the Wlds mutation.
Highlights
Mice carrying the spontaneous genetic mutation known as Wallerian degeneration slow (Wlds) 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
A more robust method for genotyping WldS mice was reported by Mi and colleagues [20] based on Pulsed Field Gel Electrophoresis (PFGE)
Quantitative PCR (QPCR) on genomic DNA for genotyping Wlds mice C57BL/6J and C57BL/Wlds mice were obtained from Harlan UK and, where required were cross-bred to obtain F1 mice, heterozygous for Wlds
Summary
Design of a novel quantitative PCR (QPCR)-based protocol for genotyping mice carrying the neuroprotective Wallerian degeneration slow (Wlds) gene. Thomas M Wishart*1,2, Stephen HF MacDonald, Philip E Chen, Michael J Shipston, Michael P Coleman, Thomas H Gillingwater and Richard R Ribchester. Address: 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 and 4Laboratory of Molecular Signalling, Babraham Institute, Babraham, Cambridge, UK. Published: 30 October 2007 Molecular Neurodegeneration 2007, 2:21 doi:10.1186/1750-1326-2-21.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
