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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 TM, Macdonald SH, Chen PE, Shipston MJ, Coleman MP, Gillingwater TH, Ribchester RR - Mol Neurodegener (2007)

Bottom Line: However, the phenotype shows strong gene-dose dependence so it is important to distinguish offspring that are homozygous or heterozygous for the mutation.We have developed a rapid, robust and efficient genotyping method for Wlds using QPCR.We have developed a QPCR genotyping method that permits rapid and effective genotyping of Wlds copy number.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Integrative Physiology, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh, UK. T.M.Wishart@ed.ac.uk.

ABSTRACT

Background: 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).

Results: 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.

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus

QPCR on genomic DNA shows clear difference in ΔCt for the 3 genotypes. A graphical representation of ΔCt between the tubulin and Wld amplicons, for animals of known genotype (N = 36, box and whisker) and animals of unknown genotype (N = 91, scatter). The areas shown in blue represent the 95% confidence limits for each particular genotype as determined from the box and whisker plots. There is a clear trend for each particular genotype.
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Figure 4: QPCR on genomic DNA shows clear difference in ΔCt for the 3 genotypes. A graphical representation of ΔCt between the tubulin and Wld amplicons, for animals of known genotype (N = 36, box and whisker) and animals of unknown genotype (N = 91, scatter). The areas shown in blue represent the 95% confidence limits for each particular genotype as determined from the box and whisker plots. There is a clear trend for each particular genotype.

Mentions: The product of a PCR for the Wlds amplicon alone run on an ethidium bromide stained gel showed that bands from each genotype could not be distinguished from each other (data not shown). However, the 2-ΔΔCT method (for more detail see Applied Biosystems website for user bulletin #2), using β-tubulin as an endogenous control and Wlds as a calibrator, allowed determination of copy number. Amplification plots for wild-type, heterozygous-Wlds and homozygous-Wlds showed clear differences for the ΔCT between each group (Figure 3 &4). The difference between β-tubulin and Wlds amplicon product at the set threshold can be used to determine Wlds genotype. A wild-type mouse gives a mean cycle difference of -0.40 (± 0.26 SD) with 95% confidence limits of -1.04 & +0.24. A mouse heterozygous for Wlds gives a mean cycle difference of 1.07 (± 0.32 SD) with 95% confidence limits of +0.97& +1.17. A mouse homozygous for Wlds gives mean cycle difference of 2.07 (± 0.30 SD) with 95% confidence limits of +01.995 & +2.158 (Figure 4).


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

Wishart TM, Macdonald SH, Chen PE, Shipston MJ, Coleman MP, Gillingwater TH, Ribchester RR - Mol Neurodegener (2007)

QPCR on genomic DNA shows clear difference in ΔCt for the 3 genotypes. A graphical representation of ΔCt between the tubulin and Wld amplicons, for animals of known genotype (N = 36, box and whisker) and animals of unknown genotype (N = 91, scatter). The areas shown in blue represent the 95% confidence limits for each particular genotype as determined from the box and whisker plots. There is a clear trend for each particular genotype.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2147001&req=5

Figure 4: QPCR on genomic DNA shows clear difference in ΔCt for the 3 genotypes. A graphical representation of ΔCt between the tubulin and Wld amplicons, for animals of known genotype (N = 36, box and whisker) and animals of unknown genotype (N = 91, scatter). The areas shown in blue represent the 95% confidence limits for each particular genotype as determined from the box and whisker plots. There is a clear trend for each particular genotype.
Mentions: The product of a PCR for the Wlds amplicon alone run on an ethidium bromide stained gel showed that bands from each genotype could not be distinguished from each other (data not shown). However, the 2-ΔΔCT method (for more detail see Applied Biosystems website for user bulletin #2), using β-tubulin as an endogenous control and Wlds as a calibrator, allowed determination of copy number. Amplification plots for wild-type, heterozygous-Wlds and homozygous-Wlds showed clear differences for the ΔCT between each group (Figure 3 &4). The difference between β-tubulin and Wlds amplicon product at the set threshold can be used to determine Wlds genotype. A wild-type mouse gives a mean cycle difference of -0.40 (± 0.26 SD) with 95% confidence limits of -1.04 & +0.24. A mouse heterozygous for Wlds gives a mean cycle difference of 1.07 (± 0.32 SD) with 95% confidence limits of +0.97& +1.17. A mouse homozygous for Wlds gives mean cycle difference of 2.07 (± 0.30 SD) with 95% confidence limits of +01.995 & +2.158 (Figure 4).

Bottom Line: However, the phenotype shows strong gene-dose dependence so it is important to distinguish offspring that are homozygous or heterozygous for the mutation.We have developed a rapid, robust and efficient genotyping method for Wlds using QPCR.We have developed a QPCR genotyping method that permits rapid and effective genotyping of Wlds copy number.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Integrative Physiology, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh, UK. T.M.Wishart@ed.ac.uk.

ABSTRACT

Background: 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).

Results: 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.

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus