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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

Primers for Wlds and β-tubulin have very similar R2 values. Standard curves for the Wlds and β-tubulin gene in a serially diluted (1 in 10) template genomic DNA sample from wild-type (C57Bl/6J). Efficient amplification was obtained for both sets of primers, as demonstrated by the slopes of linear regression of the standard curves, and good correlation coefficients. Ct is cycle threshold.
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Figure 2: Primers for Wlds and β-tubulin have very similar R2 values. Standard curves for the Wlds and β-tubulin gene in a serially diluted (1 in 10) template genomic DNA sample from wild-type (C57Bl/6J). Efficient amplification was obtained for both sets of primers, as demonstrated by the slopes of linear regression of the standard curves, and good correlation coefficients. Ct is cycle threshold.

Mentions: A serial dilution of genomic DNA from wild-type C57Bl/6J mice was used as a template for PCR to test the efficiency of amplification of the two primer pairs. The genomic DNA was diluted 1 in 10 each time and correlation coefficients were obtained for each primer pair (Figure 2). PCR efficiency was calculated by plotting the Threshold cycle (Ct) as a function of Log10 concentration of the template used (Figure 2; x-axis plotted as -Log μg DNA. See Applied Biosystems website for user bulletin #2 [22]). The slope of the trend line produced is a function of PCR efficiency, with a slope of -3.32 indicating close to 100% efficiency [22]. Standard curves suggested efficient amplification of both primer sets, as indicated by correlation coefficients and linear regression slopes. Primers against a region of Wlds and β-tubulin gave correlation coefficients of 0.9989 and 0.9928 respectively, with slopes of -3.455 for β-tubulin and -3.3647 for Wlds (Figure 2). The reactions were found to proceed similarly when primers were used both separately and together, indicating that the two separate primer pairs do not interact with each other. Once this had been demonstrated, genotyping reactions were reliably performed containing both sets of primers and probes in the same well.


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)

Primers for Wlds and β-tubulin have very similar R2 values. Standard curves for the Wlds and β-tubulin gene in a serially diluted (1 in 10) template genomic DNA sample from wild-type (C57Bl/6J). Efficient amplification was obtained for both sets of primers, as demonstrated by the slopes of linear regression of the standard curves, and good correlation coefficients. Ct is cycle threshold.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Primers for Wlds and β-tubulin have very similar R2 values. Standard curves for the Wlds and β-tubulin gene in a serially diluted (1 in 10) template genomic DNA sample from wild-type (C57Bl/6J). Efficient amplification was obtained for both sets of primers, as demonstrated by the slopes of linear regression of the standard curves, and good correlation coefficients. Ct is cycle threshold.
Mentions: A serial dilution of genomic DNA from wild-type C57Bl/6J mice was used as a template for PCR to test the efficiency of amplification of the two primer pairs. The genomic DNA was diluted 1 in 10 each time and correlation coefficients were obtained for each primer pair (Figure 2). PCR efficiency was calculated by plotting the Threshold cycle (Ct) as a function of Log10 concentration of the template used (Figure 2; x-axis plotted as -Log μg DNA. See Applied Biosystems website for user bulletin #2 [22]). The slope of the trend line produced is a function of PCR efficiency, with a slope of -3.32 indicating close to 100% efficiency [22]. Standard curves suggested efficient amplification of both primer sets, as indicated by correlation coefficients and linear regression slopes. Primers against a region of Wlds and β-tubulin gave correlation coefficients of 0.9989 and 0.9928 respectively, with slopes of -3.455 for β-tubulin and -3.3647 for Wlds (Figure 2). The reactions were found to proceed similarly when primers were used both separately and together, indicating that the two separate primer pairs do not interact with each other. Once this had been demonstrated, genotyping reactions were reliably performed containing both sets of primers and probes in the same well.

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