Developing New Tools for Detecting Germline Mutation Induction in Mice

Abstract

Identifying factors that influence germline mutation rate in animals is an important toxicological consideration. However, research in this area is limited by the lack of efficient and precise tools for characterizing germ cell mutagens in humans and model species such as mice. Thus, the purpose of my research was to develop new tools for quantifying germline mutation induction in mice. My first objective was to identify polymorphic microsatellites as a new tool for detecting changes in germline mutation frequency. Enrichment and DNA sequencing of microsatellites in closely related inbred mouse lines was used to identify polymorphic loci. The loci were used to screen family pedigrees of outbred Swiss-Webster mice in which the sires were irradiated. No mutation induction was detected using the microsatellites; however, the samples used had previously shown mutation induction at other tandem repeat loci. Failure to detect mutation induction using the microsatellites does not rule them out as a useful tool, but rather raises questions as to why induction was detected at other loci but not at microsatellites. After negative results with microsatellites, I determined if whole genome sequencing (WGS) using next-generation sequencing (NGS) technologies would be a suitable alternative for detecting germline mutation induction. WGS is more relevant than tandem repeat DNA markers to health because it examines all loci, both neutral and coding, simultaneously. However, NGS is still limited by sequencing error rates, which are high in comparison to the intergenerational mutation rate, and the high cost associated with sequencing multiple large genomes. Therefore, WGS is not yet suitable for largescale germline mutation studies. In conclusion, WGS will be an optimal tool for germline mutation studies in the near future when costs and error rates decline. WGS will need to be used to determine if there is a correlation between mutation induction at coding sequence and at neutral marker loci. If a correlation exists, then germline mutation studies using neutral markers, such as microsatellites, can continue to serve as primary tools for detecting germline mutation induction.