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.

Single-molecule PCR analysis of an unstable microsatellite for detecting mutations in sperm of mice exposed to chemical mutagens

“…Comparison of ENU-mutagenesis using tandem repeat loci versus the TGR assay at the same dose shows that the TGR assay consistently reports a higher fold increase in mutation frequency following ENU exposure [12,19,36,37]. It has been demonstrated using ENU that the mutation response of ESTRs in sperm appears to plateau at a 3-fold increase [19], whereas ENU-induced point mutations plateau at a 5-fold increase [36,14].…”

“…It has been demonstrated using ENU that the mutation response of ESTRs in sperm appears to plateau at a 3-fold increase [19], whereas ENU-induced point mutations plateau at a 5-fold increase [36,14]. Similarly, we found that the mutation induction observed at the microsatellite locus following both the ENU (2.4-fold) and BaP (2.3-fold) exposures were lower than the induced lacZ mutation frequency measured using the TGR assay (∼5-fold) in the same samples (ENU published in [16,36]; BaP unpublished data).…”

“…For the first study, treated mice received an acute dose of ENU (100 mg/kg) dissolved in phosphate buffer (pH 6, 8.71 mM Na 2 HPO 4 , 58.29 mM KH 2 PO 4 ) via oral gavage, and control animals received phosphate buffer [16,36]. Cauda epididymides were collected from three treated and three untreated males 10 weeks post-exposure (sperm exposed as spermatogonial stem cells).…”

“…By screening highly inbred strains for microsatellite polymorphisms, as a likely reflection of high mutation rate, we identify Mm2.2.1 as a sensitive marker for SM-PCR mutation detection. We test the efficacy of this SM-PCR approach by determining mutation frequencies in the sperm of mice exposed to N-ethyl-N-nitrosourea (ENU), a prototypical germ-cell mutagen tested by both the TGR [12,14,16,36] and ESTR [19,37,38] assays, and benzo(a)pyrene (BaP), a common environmental pollutant [39] following spermatogonial exposure. BaP is an established somatic-cell mutagen [39], and there is also evidence that BaP induces germ cell mutations [40][41][42], but there is currently no evidence that BaP induces tandem repeat mutations through the male germline.…”

Transgenic Rodent Gene Mutation Assay in Somatic Tissues

“…The application of transgenic rodent (TGR) models to investigate the induction of mutations in germ cells involves many considerations both in terms of the timing for sample collection and in the methods used to isolate DNA. This topic has been covered elsewhere [ 10 ].…”