Marc Beal - Academia.edu (original) (raw)
Papers by Marc Beal
Scientific Reports, 2016
De novo mutations are implicated in a variety of genetic diseases and arise primarily in the male... more De novo mutations are implicated in a variety of genetic diseases and arise primarily in the male germline. We investigated whether male germ cells have unique mechanisms for spontaneous or chemically-induced mutation relative to somatic cells using the MutaMouse model. We recovered lacZ transgenes from sperm 42 days after a 28-day exposure to benzo(a)pyrene (BaP, 100 mg/kg/day) to assess mutations arising in dividing spermatogonia. BaP caused a 3.4-fold increase in lacZ mutant frequency over controls which increased to 4.1-fold after clonal correction. We then used next generation sequencing to compare the spontaneous and BaP-induced mutation spectra in sperm and bone marrow. The spontaneous spectrum in sperm had significantly more G:C to A:T transitions and fewer mutations at A:T basepairs than bone marrow. BaP predominantly induced G:C to T:A transversions in both cell types, and both were enriched for mutations at CpG dinucleotides. However, BaP induced significantly more deletions in sperm, but more G:C to A:T transitions and G:C to C:G transversions in bone marrow. Differences in error-prone translesion DNA synthesis polymerases may underlie the observed spectrum differences between sperm and bone marrow. These findings suggest that mutations in sperm can arise via mechanisms that are unique to male germ cells.
Environmental Health Perspectives, 2016
Mosaicism, the presence of genetically distinct cell populations within an organism, has emerged ... more Mosaicism, the presence of genetically distinct cell populations within an organism, has emerged as an important contributor to disease. Mutational events occurring during embryonic development can cause mosaicism in any tissue, but the influence of environmental factors on levels of mosaicism is unclear. We investigated whether in utero exposure to the widespread environmental mutagen benzo[a]pyrene (BaP) impacts the burden and distribution of mutations in adult mice. We used the Muta™Mouse transgenic rodent model to quantify and characterize mutations in the offspring of pregnant mice exposed to BaP during postconception days 7 through 16, covering the major period of organogenesis in mice. Next-generation DNA sequencing was then used to determine the spectrum of mutations induced in adult mice that were exposed to BaP during fetal development. Mutation frequency was significantly increased in the bone marrow, liver, brain, and sperm of F1 males. Developing embryos accumulated more mutations and exhibited higher proportions of mosaicism than exposed adults, particularly in the brain. Decreased sperm count and motility revealed additional negative impacts on reproductive function of F1 males. We show that in utero exposure to environmental mutagens contributes to somatic and germline mosaicism, permanently impacting both the genetic health of the F1 and the population gene pool.
Mutation Research Fundamental and Molecular Mechanisms of Mutagenesis, Dec 10, 2011
Factors affecting the type and frequency of germline mutations in animals are of significant inte... more Factors affecting the type and frequency of germline mutations in animals are of significant interest from health and toxicology perspectives. However, studies in this field have been limited by the use of markers with low detection power or uncertain relevance to phenotype. Whole genome sequencing (WGS) is now a potential option to directly determine germline mutation type and frequency in family groups at all loci simultaneously. Medical studies have already capitalized on WGS to identify novel mutations in human families for clinical purposes, such as identifying candidate genes contributing to inherited conditions. However, WGS has not yet been used in any studies of vertebrates that aim to quantify changes in germline mutation frequency as a result of environmental factors. WGS is a promising tool for detecting mutation induction, but it is currently limited by several technical challenges. Perhaps the most pressing issue is sequencing error rates that are currently high in comparison to the intergenerational mutation frequency. Different platforms and depths of coverage currently result in a range of 10-10(3) false positives for every true mutation. In addition, the cost of WGS is still relatively high, particularly when comparing mutation frequencies among treatment groups with even moderate sample sizes. Despite these challenges, WGS offers the potential for unprecedented insight into germline mutation processes. Refinement of available tools and emergence of new technologies may be able to provide the improved accuracy and reduced costs necessary to make WGS viable in germline mutation studies in the very near future. To streamline studies, researchers may use multiple family triads per treatment group and sequence a targeted (reduced) portion of each genome with high (20-40 ×) depth of coverage. We are optimistic about the application of WGS for quantifying germline mutations, but caution researchers regarding the resource-intensive nature of the work using existing technology.
Toxicological sciences : an official journal of the Society of Toxicology, Jan 13, 2016
Although many environmental agents are established male germ cell mutagens, few are known to indu... more Although many environmental agents are established male germ cell mutagens, few are known to induce mutations in spermatogonial stem cells. Stem cell mutations are of great concern because they result in a permanent increase in the number mutations carried in sperm. We investigated mutation induction during mouse spermatogenesis following exposure to benzo(a)pyrene (BaP). Muta(TM)Mouse males were given 0, 12.5, 25, 50, or 100 mg/kg bw/day BaP for 28 days by oral gavage. Germ cells were collected from the cauda epididymis and seminiferous tubules 3 days after exposure and from cauda epididymis 42 and 70 days after exposure. This design enabled targeted investigation of effects on post-spermatogonia, dividing spermatogonia and spermatogonial stem cells, respectively. BaP increased lacZ mutant frequency (MF) in cauda sperm after exposure of dividing spermatogonia (4.2-fold at highest dose, p<0.01) and spermatogonial stem cells (2.1-fold at highest dose, p<0.01). No significant in...
BMC Genomics, 2015
The transgenic rodent mutation reporter assay provides an efficient approach to identify mutageni... more The transgenic rodent mutation reporter assay provides an efficient approach to identify mutagenic agents in vivo. A major advantage of this assay is that mutant reporter transgenes can be sequenced to provide information on the mode of action of a mutagen and to identify clonally expanded mutations. However, conventional DNA sequence analysis is laborious and expensive for long transgenes, such as lacZ (3096 bp), and is not normally implemented in routine screening. We developed a high-throughput next-generation sequencing (NGS) approach to simultaneously sequence large numbers of barcoded mutant lacZ transgenes from different animals. We collected 3872 mutants derived from the bone marrow DNA of six Muta™Mouse males exposed to the well-established mutagen benzo[a]pyrene (BaP) and six solvent-exposed controls. Mutants within animal samples were pooled, barcoded, and then sequenced using NGS. We identified 1652 mutant sequences from 1006 independent mutations that underwent clonal expansion. This deep sequencing analysis of mutation spectrum demonstrated that BaP causes primarily guanine transversions (e.g. G:C → T:A), which is highly consistent with previous studies employing Sanger sequencing. Furthermore, we identified novel mutational hotspots in the lacZ transgene that were previously uncharacterized by Sanger sequencing. Deep sequencing also allowed for an unprecedented ability to correct for clonal expansion events, improving the sensitivity of the mutation reporter assay by 50 %. These results demonstrate that the high-throughput nature and reduced costs offered by NGS provide a sensitive and fast approach for elucidating and comparing mutagenic mechanisms of various agents among tissues and enabling improved evaluation of genotoxins.
BMC genomics, Jan 19, 2015
The transgenic rodent mutation reporter assay provides an efficient approach to identify mutageni... more The transgenic rodent mutation reporter assay provides an efficient approach to identify mutagenic agents in vivo. A major advantage of this assay is that mutant reporter transgenes can be sequenced to provide information on the mode of action of a mutagen and to identify clonally expanded mutations. However, conventional DNA sequence analysis is laborious and expensive for long transgenes, such as lacZ (3096 bp), and is not normally implemented in routine screening. We developed a high-throughput next-generation sequencing (NGS) approach to simultaneously sequence large numbers of barcoded mutant lacZ transgenes from different animals. We collected 3872 mutants derived from the bone marrow DNA of six Muta™Mouse males exposed to the well-established mutagen benzo[a]pyrene (BaP) and six solvent-exposed controls. Mutants within animal samples were pooled, barcoded, and then sequenced using NGS. We identified 1652 mutant sequences from 1006 independent mutations that underwent clonal e...
Paternal Influences on Human Reproductive Success, 2013
Journal of Visualized Experiments, 2014
De novo mutations arise mostly in the male germline and may contribute to adverse health outcomes... more De novo mutations arise mostly in the male germline and may contribute to adverse health outcomes in subsequent generations. Traditional methods for assessing the induction of germ cell mutations require the use of large numbers of animals, making them impractical. As such, germ cell mutagenicity is rarely assessed during chemical testing and risk assessment. Herein, we describe an in vivo male germ cell mutation assay using a transgenic rodent model that is based on a recently approved Organisation for Economic Co-operation and Development (OECD) test guideline. This method uses an in vitro positive selection assay to measure in vivo mutations induced in a transgenic λgt10 vector bearing a reporter gene directly in the germ cells of exposed males. We further describe how the detection of mutations in the transgene recovered from germ cells can be used to characterize the stage-specific sensitivity of the various spermatogenic cell types to mutagen exposure by controlling three experimental parameters: the duration of exposure (administration time), the time between exposure and sample collection (sampling time), and the cell population collected for analysis. Because a large number of germ cells can be assayed from a single male, this method has superior sensitivity compared with traditional methods, requires fewer animals and therefore much less time and resources.
Mutation Research/Reviews in Mutation Research, 2012
Factors affecting the type and frequency of germline mutations in animals are of significant inte... more Factors affecting the type and frequency of germline mutations in animals are of significant interest from health and toxicology perspectives. However, studies in this field have been limited by the use of markers with low detection power or uncertain relevance to phenotype. Whole genome sequencing (WGS) is now a potential option to directly determine germline mutation type and frequency in family groups at all loci simultaneously. Medical studies have already capitalized on WGS to identify novel mutations in human families for clinical purposes, such as identifying candidate genes contributing to inherited conditions. However, WGS has not yet been used in any studies of vertebrates that aim to quantify changes in germline mutation frequency as a result of environmental factors. WGS is a promising tool for detecting mutation induction, but it is currently limited by several technical challenges. Perhaps the most pressing issue is sequencing error rates that are currently high in comparison to the intergenerational mutation frequency. Different platforms and depths of coverage currently result in a range of 10-10(3) false positives for every true mutation. In addition, the cost of WGS is still relatively high, particularly when comparing mutation frequencies among treatment groups with even moderate sample sizes. Despite these challenges, WGS offers the potential for unprecedented insight into germline mutation processes. Refinement of available tools and emergence of new technologies may be able to provide the improved accuracy and reduced costs necessary to make WGS viable in germline mutation studies in the very near future. To streamline studies, researchers may use multiple family triads per treatment group and sequence a targeted (reduced) portion of each genome with high (20-40 ×) depth of coverage. We are optimistic about the application of WGS for quantifying germline mutations, but caution researchers regarding the resource-intensive nature of the work using existing technology.
Environmental and Molecular Mutagenesis, 2012
Large tandem repeat DNA loci such as expanded simple tandem repeats and minisatellites are effici... more Large tandem repeat DNA loci such as expanded simple tandem repeats and minisatellites are efficient markers for detecting germline mutations; however, mutation detection using these loci can be imprecise and difficult to standardize across labs. Short-tandem repeats, such as microsatellites, offer more precise and high-throughput mutation detection, but germline mutation induction at these loci has not yet been studied in model organisms such as mice. In this study, we used microsatellite enrichment and large-scale DNA sequencing of several closely related inbred mouse lines to identify a panel of 19 polymorphic microsatellites with potentially high spontaneous mutation frequencies. We used this panel and four additional loci from other sources to quantify spontaneous mutation frequency in pedigrees of outbred Swiss-Webster mice. In addition, we also examined mutation induction in families in which sires were treated with acute doses of either 0.5 Gy or 1.0 Gy gamma-irradiation to spermatogonial stem cells. Per locus mutation frequencies ranged from 0 to 5.03 3 10 23 . Considering only the 11 loci with mutations, the mutation frequencies were: control 2.78 3 10 23 , 0.5 Gy 4.09 3 10 23 , and 1.0 Gy 1.82 3 10 23 . There were no statistically significant changes in mutation frequencies among treatment groups. Our study provides the first direct quantification of microsatellite mutation frequency in the mouse germline, but shows no evidence for mutation induction at pre-meiotic male germ cells following acute gamma-irradiation. Further work using the panel is needed to examine mutation induction at different doses of radiation, exposure durations, and stages during spermatogenesis. Environ. Mol. Mutagen. 00:000-000, 2012. V V C 2012 Wiley Periodicals, Inc.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 2015
Single-molecule PCR (SM-PCR) analysis of long and repetitive DNA sequences, known as expanded sim... more Single-molecule PCR (SM-PCR) analysis of long and repetitive DNA sequences, known as expanded simple tandem repeats (ESTRs), has been the most efficient method for studying germline mutation induction in endogenous sequences to date. However, the long length of these sequences makes mutation detection imprecise and laborious, and they have been characterized only in mice. Here, we explore the use of unstable microsatellite sequences that can be typed with high precision by capillary electrophoresis as alternative loci for detecting germline mutations. We screened 24 microsatellite loci across inbred mouse strains and identified Mm2.2.1 as the most polymorphic microsatellite locus. We then optimized SM-PCR of Mm2.2.1 to detect mutations in sperm. SM-PCR analysis of sperm from untreated B6C3F1 and Muta TM Mouse samples revealed mutation frequencies that are consistent with rates derived from family pedigree analysis (∼5 × 10 −3 ). To determine whether this locus can be used to detect chemically induced germline mutations, Muta TM Mouse males were exposed by oral gavage to a single dose of 100 mg/kg of Nethyl-N-nitrosourea (ENU) or to 100 mg/kg of benzo(a)pyrene (BaP) for 28 days alongside vehicle treated controls. Sperm were collected 10 weeks post-ENU exposure to sample sperm exposed as spermatogonial stem cells and 6 weeks post-BaP exposure to sample sperm that were dividing spermatogonia when the exposure was terminated. Both treatments resulted in a significant (approximately 2-fold) increase in mutation frequency in sperm compared to the control animals. The work establishes the utility of this microsatellite for studying mutation induction in the germ cells of mice. Because microsatellites are found in virtually every species, this approach holds promise for other organisms, including humans.
Scientific Reports, 2016
De novo mutations are implicated in a variety of genetic diseases and arise primarily in the male... more De novo mutations are implicated in a variety of genetic diseases and arise primarily in the male germline. We investigated whether male germ cells have unique mechanisms for spontaneous or chemically-induced mutation relative to somatic cells using the MutaMouse model. We recovered lacZ transgenes from sperm 42 days after a 28-day exposure to benzo(a)pyrene (BaP, 100 mg/kg/day) to assess mutations arising in dividing spermatogonia. BaP caused a 3.4-fold increase in lacZ mutant frequency over controls which increased to 4.1-fold after clonal correction. We then used next generation sequencing to compare the spontaneous and BaP-induced mutation spectra in sperm and bone marrow. The spontaneous spectrum in sperm had significantly more G:C to A:T transitions and fewer mutations at A:T basepairs than bone marrow. BaP predominantly induced G:C to T:A transversions in both cell types, and both were enriched for mutations at CpG dinucleotides. However, BaP induced significantly more deletions in sperm, but more G:C to A:T transitions and G:C to C:G transversions in bone marrow. Differences in error-prone translesion DNA synthesis polymerases may underlie the observed spectrum differences between sperm and bone marrow. These findings suggest that mutations in sperm can arise via mechanisms that are unique to male germ cells.
Environmental Health Perspectives, 2016
Mosaicism, the presence of genetically distinct cell populations within an organism, has emerged ... more Mosaicism, the presence of genetically distinct cell populations within an organism, has emerged as an important contributor to disease. Mutational events occurring during embryonic development can cause mosaicism in any tissue, but the influence of environmental factors on levels of mosaicism is unclear. We investigated whether in utero exposure to the widespread environmental mutagen benzo[a]pyrene (BaP) impacts the burden and distribution of mutations in adult mice. We used the Muta™Mouse transgenic rodent model to quantify and characterize mutations in the offspring of pregnant mice exposed to BaP during postconception days 7 through 16, covering the major period of organogenesis in mice. Next-generation DNA sequencing was then used to determine the spectrum of mutations induced in adult mice that were exposed to BaP during fetal development. Mutation frequency was significantly increased in the bone marrow, liver, brain, and sperm of F1 males. Developing embryos accumulated more mutations and exhibited higher proportions of mosaicism than exposed adults, particularly in the brain. Decreased sperm count and motility revealed additional negative impacts on reproductive function of F1 males. We show that in utero exposure to environmental mutagens contributes to somatic and germline mosaicism, permanently impacting both the genetic health of the F1 and the population gene pool.
Mutation Research Fundamental and Molecular Mechanisms of Mutagenesis, Dec 10, 2011
Factors affecting the type and frequency of germline mutations in animals are of significant inte... more Factors affecting the type and frequency of germline mutations in animals are of significant interest from health and toxicology perspectives. However, studies in this field have been limited by the use of markers with low detection power or uncertain relevance to phenotype. Whole genome sequencing (WGS) is now a potential option to directly determine germline mutation type and frequency in family groups at all loci simultaneously. Medical studies have already capitalized on WGS to identify novel mutations in human families for clinical purposes, such as identifying candidate genes contributing to inherited conditions. However, WGS has not yet been used in any studies of vertebrates that aim to quantify changes in germline mutation frequency as a result of environmental factors. WGS is a promising tool for detecting mutation induction, but it is currently limited by several technical challenges. Perhaps the most pressing issue is sequencing error rates that are currently high in comparison to the intergenerational mutation frequency. Different platforms and depths of coverage currently result in a range of 10-10(3) false positives for every true mutation. In addition, the cost of WGS is still relatively high, particularly when comparing mutation frequencies among treatment groups with even moderate sample sizes. Despite these challenges, WGS offers the potential for unprecedented insight into germline mutation processes. Refinement of available tools and emergence of new technologies may be able to provide the improved accuracy and reduced costs necessary to make WGS viable in germline mutation studies in the very near future. To streamline studies, researchers may use multiple family triads per treatment group and sequence a targeted (reduced) portion of each genome with high (20-40 ×) depth of coverage. We are optimistic about the application of WGS for quantifying germline mutations, but caution researchers regarding the resource-intensive nature of the work using existing technology.
Toxicological sciences : an official journal of the Society of Toxicology, Jan 13, 2016
Although many environmental agents are established male germ cell mutagens, few are known to indu... more Although many environmental agents are established male germ cell mutagens, few are known to induce mutations in spermatogonial stem cells. Stem cell mutations are of great concern because they result in a permanent increase in the number mutations carried in sperm. We investigated mutation induction during mouse spermatogenesis following exposure to benzo(a)pyrene (BaP). Muta(TM)Mouse males were given 0, 12.5, 25, 50, or 100 mg/kg bw/day BaP for 28 days by oral gavage. Germ cells were collected from the cauda epididymis and seminiferous tubules 3 days after exposure and from cauda epididymis 42 and 70 days after exposure. This design enabled targeted investigation of effects on post-spermatogonia, dividing spermatogonia and spermatogonial stem cells, respectively. BaP increased lacZ mutant frequency (MF) in cauda sperm after exposure of dividing spermatogonia (4.2-fold at highest dose, p<0.01) and spermatogonial stem cells (2.1-fold at highest dose, p<0.01). No significant in...
BMC Genomics, 2015
The transgenic rodent mutation reporter assay provides an efficient approach to identify mutageni... more The transgenic rodent mutation reporter assay provides an efficient approach to identify mutagenic agents in vivo. A major advantage of this assay is that mutant reporter transgenes can be sequenced to provide information on the mode of action of a mutagen and to identify clonally expanded mutations. However, conventional DNA sequence analysis is laborious and expensive for long transgenes, such as lacZ (3096 bp), and is not normally implemented in routine screening. We developed a high-throughput next-generation sequencing (NGS) approach to simultaneously sequence large numbers of barcoded mutant lacZ transgenes from different animals. We collected 3872 mutants derived from the bone marrow DNA of six Muta™Mouse males exposed to the well-established mutagen benzo[a]pyrene (BaP) and six solvent-exposed controls. Mutants within animal samples were pooled, barcoded, and then sequenced using NGS. We identified 1652 mutant sequences from 1006 independent mutations that underwent clonal expansion. This deep sequencing analysis of mutation spectrum demonstrated that BaP causes primarily guanine transversions (e.g. G:C → T:A), which is highly consistent with previous studies employing Sanger sequencing. Furthermore, we identified novel mutational hotspots in the lacZ transgene that were previously uncharacterized by Sanger sequencing. Deep sequencing also allowed for an unprecedented ability to correct for clonal expansion events, improving the sensitivity of the mutation reporter assay by 50 %. These results demonstrate that the high-throughput nature and reduced costs offered by NGS provide a sensitive and fast approach for elucidating and comparing mutagenic mechanisms of various agents among tissues and enabling improved evaluation of genotoxins.
BMC genomics, Jan 19, 2015
The transgenic rodent mutation reporter assay provides an efficient approach to identify mutageni... more The transgenic rodent mutation reporter assay provides an efficient approach to identify mutagenic agents in vivo. A major advantage of this assay is that mutant reporter transgenes can be sequenced to provide information on the mode of action of a mutagen and to identify clonally expanded mutations. However, conventional DNA sequence analysis is laborious and expensive for long transgenes, such as lacZ (3096 bp), and is not normally implemented in routine screening. We developed a high-throughput next-generation sequencing (NGS) approach to simultaneously sequence large numbers of barcoded mutant lacZ transgenes from different animals. We collected 3872 mutants derived from the bone marrow DNA of six Muta™Mouse males exposed to the well-established mutagen benzo[a]pyrene (BaP) and six solvent-exposed controls. Mutants within animal samples were pooled, barcoded, and then sequenced using NGS. We identified 1652 mutant sequences from 1006 independent mutations that underwent clonal e...
Paternal Influences on Human Reproductive Success, 2013
Journal of Visualized Experiments, 2014
De novo mutations arise mostly in the male germline and may contribute to adverse health outcomes... more De novo mutations arise mostly in the male germline and may contribute to adverse health outcomes in subsequent generations. Traditional methods for assessing the induction of germ cell mutations require the use of large numbers of animals, making them impractical. As such, germ cell mutagenicity is rarely assessed during chemical testing and risk assessment. Herein, we describe an in vivo male germ cell mutation assay using a transgenic rodent model that is based on a recently approved Organisation for Economic Co-operation and Development (OECD) test guideline. This method uses an in vitro positive selection assay to measure in vivo mutations induced in a transgenic λgt10 vector bearing a reporter gene directly in the germ cells of exposed males. We further describe how the detection of mutations in the transgene recovered from germ cells can be used to characterize the stage-specific sensitivity of the various spermatogenic cell types to mutagen exposure by controlling three experimental parameters: the duration of exposure (administration time), the time between exposure and sample collection (sampling time), and the cell population collected for analysis. Because a large number of germ cells can be assayed from a single male, this method has superior sensitivity compared with traditional methods, requires fewer animals and therefore much less time and resources.
Mutation Research/Reviews in Mutation Research, 2012
Factors affecting the type and frequency of germline mutations in animals are of significant inte... more Factors affecting the type and frequency of germline mutations in animals are of significant interest from health and toxicology perspectives. However, studies in this field have been limited by the use of markers with low detection power or uncertain relevance to phenotype. Whole genome sequencing (WGS) is now a potential option to directly determine germline mutation type and frequency in family groups at all loci simultaneously. Medical studies have already capitalized on WGS to identify novel mutations in human families for clinical purposes, such as identifying candidate genes contributing to inherited conditions. However, WGS has not yet been used in any studies of vertebrates that aim to quantify changes in germline mutation frequency as a result of environmental factors. WGS is a promising tool for detecting mutation induction, but it is currently limited by several technical challenges. Perhaps the most pressing issue is sequencing error rates that are currently high in comparison to the intergenerational mutation frequency. Different platforms and depths of coverage currently result in a range of 10-10(3) false positives for every true mutation. In addition, the cost of WGS is still relatively high, particularly when comparing mutation frequencies among treatment groups with even moderate sample sizes. Despite these challenges, WGS offers the potential for unprecedented insight into germline mutation processes. Refinement of available tools and emergence of new technologies may be able to provide the improved accuracy and reduced costs necessary to make WGS viable in germline mutation studies in the very near future. To streamline studies, researchers may use multiple family triads per treatment group and sequence a targeted (reduced) portion of each genome with high (20-40 ×) depth of coverage. We are optimistic about the application of WGS for quantifying germline mutations, but caution researchers regarding the resource-intensive nature of the work using existing technology.
Environmental and Molecular Mutagenesis, 2012
Large tandem repeat DNA loci such as expanded simple tandem repeats and minisatellites are effici... more Large tandem repeat DNA loci such as expanded simple tandem repeats and minisatellites are efficient markers for detecting germline mutations; however, mutation detection using these loci can be imprecise and difficult to standardize across labs. Short-tandem repeats, such as microsatellites, offer more precise and high-throughput mutation detection, but germline mutation induction at these loci has not yet been studied in model organisms such as mice. In this study, we used microsatellite enrichment and large-scale DNA sequencing of several closely related inbred mouse lines to identify a panel of 19 polymorphic microsatellites with potentially high spontaneous mutation frequencies. We used this panel and four additional loci from other sources to quantify spontaneous mutation frequency in pedigrees of outbred Swiss-Webster mice. In addition, we also examined mutation induction in families in which sires were treated with acute doses of either 0.5 Gy or 1.0 Gy gamma-irradiation to spermatogonial stem cells. Per locus mutation frequencies ranged from 0 to 5.03 3 10 23 . Considering only the 11 loci with mutations, the mutation frequencies were: control 2.78 3 10 23 , 0.5 Gy 4.09 3 10 23 , and 1.0 Gy 1.82 3 10 23 . There were no statistically significant changes in mutation frequencies among treatment groups. Our study provides the first direct quantification of microsatellite mutation frequency in the mouse germline, but shows no evidence for mutation induction at pre-meiotic male germ cells following acute gamma-irradiation. Further work using the panel is needed to examine mutation induction at different doses of radiation, exposure durations, and stages during spermatogenesis. Environ. Mol. Mutagen. 00:000-000, 2012. V V C 2012 Wiley Periodicals, Inc.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 2015
Single-molecule PCR (SM-PCR) analysis of long and repetitive DNA sequences, known as expanded sim... more Single-molecule PCR (SM-PCR) analysis of long and repetitive DNA sequences, known as expanded simple tandem repeats (ESTRs), has been the most efficient method for studying germline mutation induction in endogenous sequences to date. However, the long length of these sequences makes mutation detection imprecise and laborious, and they have been characterized only in mice. Here, we explore the use of unstable microsatellite sequences that can be typed with high precision by capillary electrophoresis as alternative loci for detecting germline mutations. We screened 24 microsatellite loci across inbred mouse strains and identified Mm2.2.1 as the most polymorphic microsatellite locus. We then optimized SM-PCR of Mm2.2.1 to detect mutations in sperm. SM-PCR analysis of sperm from untreated B6C3F1 and Muta TM Mouse samples revealed mutation frequencies that are consistent with rates derived from family pedigree analysis (∼5 × 10 −3 ). To determine whether this locus can be used to detect chemically induced germline mutations, Muta TM Mouse males were exposed by oral gavage to a single dose of 100 mg/kg of Nethyl-N-nitrosourea (ENU) or to 100 mg/kg of benzo(a)pyrene (BaP) for 28 days alongside vehicle treated controls. Sperm were collected 10 weeks post-ENU exposure to sample sperm exposed as spermatogonial stem cells and 6 weeks post-BaP exposure to sample sperm that were dividing spermatogonia when the exposure was terminated. Both treatments resulted in a significant (approximately 2-fold) increase in mutation frequency in sperm compared to the control animals. The work establishes the utility of this microsatellite for studying mutation induction in the germ cells of mice. Because microsatellites are found in virtually every species, this approach holds promise for other organisms, including humans.