title: Single-molecule PCR analysis of an unstable microsatellite for detecting mutations in sperm of mice exposed to chemical mutagens.
abstract: 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(™)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(™)Mouse males were exposed by oral gavage to a single dose of 100mg/kg of N-ethyl-N-nitrosourea (ENU) or to 100mg/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.
authors: Carleton University, Ottawa, Ontario K1S 5B6, Canada; Environmental Health Science and Research Bureau, Healthy Environments and Consumer Safety Branch, Health Canada, Ottawa, Ontario K1A 0K9, Canada. Electronic address: marc.beal@hc-sc.gc.ca.
authors: Marc A
authors: MA
authors: Beal
authors: Environmental Health Science and Research Bureau, Healthy Environments and Consumer Safety Branch, Health Canada, Ottawa, Ontario K1A 0K9, Canada. Electronic address: andrea.rowan-carroll@hc-sc.gc.ca.
authors: Andrea
authors: A
authors: Rowan-Carroll
authors: Environmental Health Science and Research Bureau, Healthy Environments and Consumer Safety Branch, Health Canada, Ottawa, Ontario K1A 0K9, Canada. Electronic address: caleigh.campbell@hotmail.com.
authors: Caleigh
authors: C
authors: Campbell
authors: Environmental Health Science and Research Bureau, Healthy Environments and Consumer Safety Branch, Health Canada, Ottawa, Ontario K1A 0K9, Canada. Electronic address: andrew.williams@hc-sc.gc.ca.
authors: Andrew
authors: A
authors: Williams
authors: University of Regina, Regina, Saskatchewan S4S 0A2, Canada. Electronic address: chris.somers@uregina.ca.
authors: Christopher M
authors: CM
authors: Somers
authors: Environmental Health Science and Research Bureau, Healthy Environments and Consumer Safety Branch, Health Canada, Ottawa, Ontario K1A 0K9, Canada. Electronic address: francesco.marchetti@hc-sc.gc.ca.
authors: Francesco
authors: F
authors: Marchetti
authors: Environmental Health Science and Research Bureau, Healthy Environments and Consumer Safety Branch, Health Canada, Ottawa, Ontario K1A 0K9, Canada. Electronic address: carole.yauk@hc-sc.gc.ca.
authors: Carole L
authors: CL
authors: Yauk
mesh_terms: D000818:Animals; D001564:Benzo(a)pyrene / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D005260:Female; D056426:Genetic Loci; D018095:Germ-Line Mutation; D008297:Male; D051379:Mice; D053842:Microsatellite Instability / Q000187:drug effects; D018895:Microsatellite Repeats / Q000235:genetics*; D016296:Mutagenesis / Q000187:drug effects*; D009153:Mutagens / Q000378:metabolism / Q000633:toxicity*; D016133:Polymerase Chain Reaction / Q000379:methods*; D013093:Spermatogonia
chemical_list: D009153:Mutagens; D001564:Benzo(a)pyrene
keywords: Germline mutation; Microsatellite; Mouse; Single-molecule PCR; Sperm