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A random mutation capture assay to detect genomic point mutations in mouse tissue.

Wright JH, Modjeski KL, Bielas JH, Preston BD, Fausto N, Loeb LA, Campbell JS - Nucleic Acids Res. (2011)

Bottom Line: We analyzed mutation frequencies from the liver tissue of animals with a mutation within the intrinsic exonuclease domains of the two major DNA polymerases, δ and ε.These mice exhibited significantly higher mutation frequencies than did wild-type animals.As RMC does not require analysis of a particular gene, simultaneous analysis of mutation frequency at multiple genetic loci is feasible.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Washington and Department of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, USA. jhw5@uw.edu

ABSTRACT
Herein, a detailed protocol for a random mutation capture (RMC) assay to measure nuclear point mutation frequency in mouse tissue is described. This protocol is a simplified version of the original method developed for human tissue that is easier to perform, yet retains a high sensitivity of detection. In contrast to assays relying on phenotypic selection of reporter genes in transgenic mice, the RMC assay allows direct detection of mutations in endogenous genes in any mouse strain. Measuring mutation frequency within an intron of a transcribed gene, we show this assay to be highly reproducible. We analyzed mutation frequencies from the liver tissue of animals with a mutation within the intrinsic exonuclease domains of the two major DNA polymerases, δ and ε. These mice exhibited significantly higher mutation frequencies than did wild-type animals. A comparison with a previous analysis of these genotypes in Big Blue mice revealed the RMC assay to be more sensitive than the Big Blue assay for this application. As RMC does not require analysis of a particular gene, simultaneous analysis of mutation frequency at multiple genetic loci is feasible. This assay provides a versatile alternative to transgenic mouse models for the study of mutagenesis in vivo.

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Relative mutation frequency for individual mice from Polee/e, Pold1e/e and wild-type C57BL/6 genotypes. Mice with different genotypes are shown in different colors; Polee/e are black, Pold1e/e in gray and wild type in light gray. Each bar represents data for an individual mouse, which was assigned a ‘code’ at necropsy to blind the experimenter to genotype during analysis. Mouse names are indicated below each datum bar, along with the mutation frequency, the number of sequence-verified mutations identified and the total number of base pairs (in millions) screened for that individual mouse.
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Figure 6: Relative mutation frequency for individual mice from Polee/e, Pold1e/e and wild-type C57BL/6 genotypes. Mice with different genotypes are shown in different colors; Polee/e are black, Pold1e/e in gray and wild type in light gray. Each bar represents data for an individual mouse, which was assigned a ‘code’ at necropsy to blind the experimenter to genotype during analysis. Mouse names are indicated below each datum bar, along with the mutation frequency, the number of sequence-verified mutations identified and the total number of base pairs (in millions) screened for that individual mouse.

Mentions: The median mutation frequency for these mice was 5.3 × 10−7 mutations/bp, with >10-fold variation in frequency between individual mice. These 9-month-old mice had significantly higher mutation frequencies than did other C57BL/6 mice at 2 months of age shown in Figure 6 and described in the following section. These results suggest that mice accumulate random mutations with age as has been shown previously using other methods (20,21). Based on these results, we recommend that at least six mice be tested per cohort and that the cohorts be age matched.Figure 6.


A random mutation capture assay to detect genomic point mutations in mouse tissue.

Wright JH, Modjeski KL, Bielas JH, Preston BD, Fausto N, Loeb LA, Campbell JS - Nucleic Acids Res. (2011)

Relative mutation frequency for individual mice from Polee/e, Pold1e/e and wild-type C57BL/6 genotypes. Mice with different genotypes are shown in different colors; Polee/e are black, Pold1e/e in gray and wild type in light gray. Each bar represents data for an individual mouse, which was assigned a ‘code’ at necropsy to blind the experimenter to genotype during analysis. Mouse names are indicated below each datum bar, along with the mutation frequency, the number of sequence-verified mutations identified and the total number of base pairs (in millions) screened for that individual mouse.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3113589&req=5

Figure 6: Relative mutation frequency for individual mice from Polee/e, Pold1e/e and wild-type C57BL/6 genotypes. Mice with different genotypes are shown in different colors; Polee/e are black, Pold1e/e in gray and wild type in light gray. Each bar represents data for an individual mouse, which was assigned a ‘code’ at necropsy to blind the experimenter to genotype during analysis. Mouse names are indicated below each datum bar, along with the mutation frequency, the number of sequence-verified mutations identified and the total number of base pairs (in millions) screened for that individual mouse.
Mentions: The median mutation frequency for these mice was 5.3 × 10−7 mutations/bp, with >10-fold variation in frequency between individual mice. These 9-month-old mice had significantly higher mutation frequencies than did other C57BL/6 mice at 2 months of age shown in Figure 6 and described in the following section. These results suggest that mice accumulate random mutations with age as has been shown previously using other methods (20,21). Based on these results, we recommend that at least six mice be tested per cohort and that the cohorts be age matched.Figure 6.

Bottom Line: We analyzed mutation frequencies from the liver tissue of animals with a mutation within the intrinsic exonuclease domains of the two major DNA polymerases, δ and ε.These mice exhibited significantly higher mutation frequencies than did wild-type animals.As RMC does not require analysis of a particular gene, simultaneous analysis of mutation frequency at multiple genetic loci is feasible.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Washington and Department of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, USA. jhw5@uw.edu

ABSTRACT
Herein, a detailed protocol for a random mutation capture (RMC) assay to measure nuclear point mutation frequency in mouse tissue is described. This protocol is a simplified version of the original method developed for human tissue that is easier to perform, yet retains a high sensitivity of detection. In contrast to assays relying on phenotypic selection of reporter genes in transgenic mice, the RMC assay allows direct detection of mutations in endogenous genes in any mouse strain. Measuring mutation frequency within an intron of a transcribed gene, we show this assay to be highly reproducible. We analyzed mutation frequencies from the liver tissue of animals with a mutation within the intrinsic exonuclease domains of the two major DNA polymerases, δ and ε. These mice exhibited significantly higher mutation frequencies than did wild-type animals. A comparison with a previous analysis of these genotypes in Big Blue mice revealed the RMC assay to be more sensitive than the Big Blue assay for this application. As RMC does not require analysis of a particular gene, simultaneous analysis of mutation frequency at multiple genetic loci is feasible. This assay provides a versatile alternative to transgenic mouse models for the study of mutagenesis in vivo.

Show MeSH
Related in: MedlinePlus