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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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Comparison of RMC analysis of mutation frequency in Trp53 intron IV with analysis of frequency in the cII transgene using Big Blue mice. (a) RMC data from the 6- to 8-week-old mice with the different genotypes are shown. Frequencies for individual mice are presented as points around the median value that is indicated as a horizontal line. Median values are as follows: 0.0 for wild type, 29.8 for Polee/e and 2.1 for Pold1e/e. The median mutation frequencies were found to be statistically different between both mutator mouse genotypes and wild type: wild type versus Polee/e, P = 0.003; wild type versus Pold1e/e, P = 0.009 and Polee/e versus Pold1e/e, P = 0.001. (b) Big Blue mouse data from a previous study (12) on 6- to 8-week-old mice of the different genotypes. Mutation frequencies measured in four different tissues are shown: small intestine (squares), lung (diamonds), thymus (triangles), bone marrow (upside down triangles). The three bars on the left show the effect of a mutation in DNA polymerase epsilon, and the three bars on the left show the effects of a mutation in DNA polymerase delta.
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Figure 7: Comparison of RMC analysis of mutation frequency in Trp53 intron IV with analysis of frequency in the cII transgene using Big Blue mice. (a) RMC data from the 6- to 8-week-old mice with the different genotypes are shown. Frequencies for individual mice are presented as points around the median value that is indicated as a horizontal line. Median values are as follows: 0.0 for wild type, 29.8 for Polee/e and 2.1 for Pold1e/e. The median mutation frequencies were found to be statistically different between both mutator mouse genotypes and wild type: wild type versus Polee/e, P = 0.003; wild type versus Pold1e/e, P = 0.009 and Polee/e versus Pold1e/e, P = 0.001. (b) Big Blue mouse data from a previous study (12) on 6- to 8-week-old mice of the different genotypes. Mutation frequencies measured in four different tissues are shown: small intestine (squares), lung (diamonds), thymus (triangles), bone marrow (upside down triangles). The three bars on the left show the effect of a mutation in DNA polymerase epsilon, and the three bars on the left show the effects of a mutation in DNA polymerase delta.

Mentions: Cohorts of six to seven mice, 6–8 weeks of age, were sacrificed from Polee/e and Pold1e/e colonies along with wild-type littermates. DNA was isolated from the left lobe of the liver, digested with TaqαI and diluted for qPCR analysis. For each sample, greater than or equal to four million base pairs were screened with mutant-specific primers. Results of this analysis are shown in Figure 6. Each bar represents the mutation frequency calculated for the individual mice. Multiple mutations were detected in all the Polee/e mice and in six out of seven Pold1e/e mice. These high mutation frequencies in the mutant mice were in sharp contrast to wild-type mice where one mutation was detected in only a single individual. Thus, elimination of proofreading activity from either of the major DNA polymerases caused a significant increase in mutation frequency within the RMC target TaqI site (Figures 6 and 7). The majority of the wild-type mice had no mutations identified giving them mutation frequencies of zero. We included less-than values shown at the bottom of Figure 6 which reflect the number of total base pairs screened for each mouse. In order to obtain a background mutation frequency value for a wild-type mouse, 55 million base pairs were screened from one sample until a mutation was identified. The calculated mutation frequency for that one wild-type mouse was 9 × 10−9 mutations/bp.Figure 7.


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)

Comparison of RMC analysis of mutation frequency in Trp53 intron IV with analysis of frequency in the cII transgene using Big Blue mice. (a) RMC data from the 6- to 8-week-old mice with the different genotypes are shown. Frequencies for individual mice are presented as points around the median value that is indicated as a horizontal line. Median values are as follows: 0.0 for wild type, 29.8 for Polee/e and 2.1 for Pold1e/e. The median mutation frequencies were found to be statistically different between both mutator mouse genotypes and wild type: wild type versus Polee/e, P = 0.003; wild type versus Pold1e/e, P = 0.009 and Polee/e versus Pold1e/e, P = 0.001. (b) Big Blue mouse data from a previous study (12) on 6- to 8-week-old mice of the different genotypes. Mutation frequencies measured in four different tissues are shown: small intestine (squares), lung (diamonds), thymus (triangles), bone marrow (upside down triangles). The three bars on the left show the effect of a mutation in DNA polymerase epsilon, and the three bars on the left show the effects of a mutation in DNA polymerase delta.
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Figure 7: Comparison of RMC analysis of mutation frequency in Trp53 intron IV with analysis of frequency in the cII transgene using Big Blue mice. (a) RMC data from the 6- to 8-week-old mice with the different genotypes are shown. Frequencies for individual mice are presented as points around the median value that is indicated as a horizontal line. Median values are as follows: 0.0 for wild type, 29.8 for Polee/e and 2.1 for Pold1e/e. The median mutation frequencies were found to be statistically different between both mutator mouse genotypes and wild type: wild type versus Polee/e, P = 0.003; wild type versus Pold1e/e, P = 0.009 and Polee/e versus Pold1e/e, P = 0.001. (b) Big Blue mouse data from a previous study (12) on 6- to 8-week-old mice of the different genotypes. Mutation frequencies measured in four different tissues are shown: small intestine (squares), lung (diamonds), thymus (triangles), bone marrow (upside down triangles). The three bars on the left show the effect of a mutation in DNA polymerase epsilon, and the three bars on the left show the effects of a mutation in DNA polymerase delta.
Mentions: Cohorts of six to seven mice, 6–8 weeks of age, were sacrificed from Polee/e and Pold1e/e colonies along with wild-type littermates. DNA was isolated from the left lobe of the liver, digested with TaqαI and diluted for qPCR analysis. For each sample, greater than or equal to four million base pairs were screened with mutant-specific primers. Results of this analysis are shown in Figure 6. Each bar represents the mutation frequency calculated for the individual mice. Multiple mutations were detected in all the Polee/e mice and in six out of seven Pold1e/e mice. These high mutation frequencies in the mutant mice were in sharp contrast to wild-type mice where one mutation was detected in only a single individual. Thus, elimination of proofreading activity from either of the major DNA polymerases caused a significant increase in mutation frequency within the RMC target TaqI site (Figures 6 and 7). The majority of the wild-type mice had no mutations identified giving them mutation frequencies of zero. We included less-than values shown at the bottom of Figure 6 which reflect the number of total base pairs screened for each mouse. In order to obtain a background mutation frequency value for a wild-type mouse, 55 million base pairs were screened from one sample until a mutation was identified. The calculated mutation frequency for that one wild-type mouse was 9 × 10−9 mutations/bp.Figure 7.

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