title: Transgenic rodent assay for quantifying male germ cell mutant frequency.
abstract: 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.
authors.affiliation: Environmental Health Science and Research Bureau, Health Canada, Environmental Health Centre.
authors.forename: Jason M
authors.initials: JM
authors.lastname: O'Brien
authors.affiliation: Environmental Health Science and Research Bureau, Health Canada, Environmental Health Centre.
authors.forename: Marc A
authors.initials: MA
authors.lastname: Beal
authors.affiliation: Environmental Health Science and Research Bureau, Health Canada, Environmental Health Centre.
authors.forename: John D
authors.initials: JD
authors.lastname: Gingerich
authors.affiliation: Environmental Health Science and Research Bureau, Health Canada, Environmental Health Centre.
authors.forename: Lynda
authors.initials: L
authors.lastname: Soper
authors.affiliation: Environmental Health Science and Research Bureau, Health Canada, Environmental Health Centre.
authors.forename: George R
authors.initials: GR
authors.lastname: Douglas
authors.affiliation: Environmental Health Science and Research Bureau, Health Canada, Environmental Health Centre.
authors.forename: Carole L
authors.initials: CL
authors.lastname: Yauk
authors.affiliation: Environmental Health Science and Research Bureau, Health Canada, Environmental Health Centre; francesco.marchetti@hc-sc.gc.ca.
authors.forename: Francesco
authors.initials: F
authors.lastname: Marchetti
mesh_terms: D000818:Animals; D004252:DNA Mutational Analysis / Q000379:methods*; D017930:Genes, Reporter; D018095:Germ-Line Mutation; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D059645:Mutation Rate; D013094:Spermatozoa / Q000166:cytology / Q000502:physiology*