Articles by Marc A. Beal in JoVE
Transgenic Rodent Assay for Quantifying Male Germ Cell Mutant Frequency Jason M. O'Brien1, Marc A. Beal1, John D. Gingerich1, Lynda Soper1, George R. Douglas1, Carole L. Yauk1, Francesco Marchetti1 1Environmental Health Science and Research Bureau, Health Canada, Environmental Health Centre De novo mutations in the male germline may contribute to adverse health outcomes in subsequent generations. Here we describe a protocol for the use of a transgenic rodent model for quantifying mutations in male germ cells induced by environmental agents.
Other articles by Marc A. Beal on PubMed
Whole Genome Sequencing for Quantifying Germline Mutation Frequency in Humans and Model Species: Cautious Optimism Mutation Research. Apr-Jun, 2012 | Pubmed ID: 22178956 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.
Characterization of Unstable Microsatellites in Mice: No Evidence for Germline Mutation Induction Following Gamma-radiation Exposure Environmental and Molecular Mutagenesis. Oct, 2012 | Pubmed ID: 22930577 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 × 10(-3). Considering only the 11 loci with mutations, the mutation frequencies were: control 2.78 × 10(-3), 0.5 Gy 4.09 × 10(-3), and 1.0 Gy 1.82 × 10(-3). 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.