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

ABSTRACT: 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. 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).We 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.We 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.

SUBMITTER: Wishart TM

PROVIDER: S-EPMC2147001 | biostudies-literature | 2007 Oct

REPOSITORIES: biostudies-literature

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

Wishart Thomas M TM Macdonald Stephen Hf SH Chen Philip E PE Shipston Michael J MJ Coleman Michael P MP Gillingwater Thomas H TH Ribchester Richard R RR

Molecular neurodegeneration 20071030

<h4>Background</h4>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. 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 ...[more]

PMID: 17971231

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Project description:Abstract: Wallerian degeneration (WD) is a process of autonomous distal degeneration of axons upon injury. Macrophages (MP) of the peripheral nervous system (PNS) are main cellular agent controlling this process. Some evidences suggest that resident PNS-MP along with MP of hematogenous origin may be involved but whether these two subsets exert distinct functions is unknown. Combining MP-designed fluorescent reporters mice, and coherent anti-stoke raman scattering (CARS) imaging of the sciatic nerve, we deciphered the spatio-temporal choreography of resident and recently recruited MP after injury and unveiled distinct functions of these subsets with recruited MP responsible of efficient myelin stripping and clearance while resident MP were involved in axonal regrowth. This work provides clues to tackle selectively cellular processes involved in neurodegenerative diseases. Methods: (relevant for this GEO dataset): RNAseq of sciatic nerve macrophages from mice after CCI: Sorted cells were lysed in 100µl of RA1/TCEP buffer (NucleoSpin RNA XS, Macherey-Nagel), snap frozen in liquid nitrogen and stored at -80C until RNA extraction. All samples were processed in parallel and RNA extraction (without Carrier RNA but with on-column DNase treatment) was performed according to manufacturer's instructions (NucleoSpin RNA XS, Macherey-Nagel). RNA was eluted in RNAse-free water. Preparation of cDNA libraries for RNAseq was done using the SmartSeq method according to manufacturer's instructions (SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing, Clontech/TaKaRa). Due to the low number of cells, the total amount of eluted RNA was used as starting material for reverse transcription, followed by 18 cycles of pre-amplification. 1ng of cDNA were used for RNAseq sequencing library preparation, according to manufacturer's instructions (Nextera XT DNA Library Preparation, Illumina). Final samples pooled library prep were sequenced on a Nextseq 500 ILLUMINA with MidOutPut cartridge (2x130Millions of 75 bases reads) with 2 runs (4plex and 5plex), corresponding to 2x30Millions of (paired-end) reads per sample after demultiplexing.

Dataset Information

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

Publications

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

Similar Datasets

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