Response to the commentary article: Comparison of mutation frequencies obtained using transgenes and the specific-locus mutation system in male mouse-germ cells (original) (raw)
Related papers
Transgenic Rodent Assay for Quantifying Male Germ Cell Mutant Frequency
Journal of Visualized Experiments, 2014
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.
Mutation spectral changes in spermatogenic cells obtained from old mice
DNA Repair, 2004
Male reproductive health is compromised with increased paternal age, due at least in part, to an increased frequency of de novo germline mutations. Because of technical and sample limitations, there is a dearth of empirical information on the mechanism(s) that mediate this age-related increase in mutant frequency. To study this phenomenon, investigators have used as a model system a transgenic mouse strain that carries a lacI mutagenesis reporter transgene. This transgene displays a paternal age effect and overcomes many of the technical difficulties that have inhibited experimental analyses of age-related changes in the male germline. In this study, approximately 300 mutant lacI transgenes were recovered from defined spermatogenic cell types obtained from various aged lacI transgenic mice and sequenced. The spectrum representing mutations from spermatogenic cells of old mice revealed an increased prevalence of transversions compared to spectra for young and middle-aged mice. Five mutation hotspots were identified in spectra for spermatogenic cells from young and middle-aged mice, but no hotspots were identified in the spectrum for spermatogenic cells from old mice. These results suggest that the challenges to germline DNA change as the animal ages and that the increased mutant frequency observed with increased paternal age is not simply a greater accumulation of mutagenic events characteristic of spermatogenic cells from the young animal.
Mutation frequency declines during spermatogenesis in young mice but increases in old mice
Proceedings of the National Academy of Sciences, 1998
Five percent of live-born human offspring will have a genetic disorder. Of these, 20% are because of germ-line de novo mutations. Several genetic diseases, such as neurofibromatosis and Duchenne muscular dystrophy, are associated with a high percentage of de novo germ-line mutations. Until recently, a direct analysis of spontaneous mutation frequencies in mammalian germ cells has been prevented by technical limitations. We have measured spontaneous mutation frequencies in a lacI transgene by using enriched populations of specific spermatogenic cell types. Similar to previously published results, we observed a lower mutation frequency for seminiferous tubule cell preparations, which contain all stages of spermatogenesis, relative to somatic tissues. We made the unexpected observation of a decline in mutation frequency during spermatogenesis, such that the mutation frequencies of type B spermatogonia and all subsequent stages of spermatogenesis are lower than the frequency for primitive type A spermatogonia. In addition, spermatogenic cells from old mice have significantly increased mutation frequencies compared with spermatogenic cells from young or middle-aged mice. Finally, the mutation frequency was observed to increase during spermiogenesis in postreplicative cell types when spermatogenic cells were obtained from old mice.
Mutagenesis Is Elevated in Male Germ Cells Obtained from DNA Polymerase-beta Heterozygous Mice
Biology of Reproduction, 2008
Gametes carry the DNA that will direct the development of the next generation. By compromising genetic integrity, DNA damage and mutagenesis threaten the ability of gametes to fulfill their biological function. DNA repair pathways function in germ cells and serve to ameliorate much DNA damage and prevent mutagenesis. High base excision repair (BER) activity is documented for spermatogenic cells. DNA polymerase-beta (POLB) is required for the short-patch BER pathway. Because mice homozygous null for the Polb gene die soon after birth, mice heterozygous for Polb were used to examine the extent to which POLB contributes to maintaining spermatogenic genomic integrity in vivo. POLB protein levels were reduced only in mixed spermatogenic cells. In vitro short-patch BER activity assays revealed that spermatogenic cell nuclear extracts obtained from Polb heterozygous mice had one third the BER activity of age-matched control mice. Polb heterozygosity had no effect on the BER activities of somatic tissues tested. The Polb heterozygous mouse line was crossed with the lacI transgenic Big Blue mouse line to assess mutant frequency. The spontaneous mutant frequency for mixed spermatogenic cells prepared from Polb heterozygous mice was 2-fold greater than that of wild-type controls, but no significant effect was found among the somatic tissues tested. These results demonstrate that normal POLB abundance is necessary for normal BER activity, which is critical in maintaining a low germline mutant frequency. Notably, spermatogenic cells respond differently than somatic cells to Polb haploinsufficiency.
A determination of the frequency of gene conversion in unmanipulated mouse sperm
Proceedings of the National Academy of Sciences, 1994
Gene conversion, sometimes also called micro gene conversion or gene conversion-like events, has been proposed to act on a number of genes in higher eukaryotes, such as gamma-globin, beta-tubulin, major urinary protein, and amyloid A genes. In the immune system, immunoglobulin genes and major histocompatibility complex class I and class II genes have been implicated. The notion that integral segments of DNA have been transferred from one gene to another in these cases has, however, met considerable resistance. We have devised a PCR assay detecting only the molecule that results if the E beta d-derived fragment analogous to that introduced in the bm12 mutation is transferred to the A beta k gene. We have proceeded to analyze sperm from the F1 cross C3H/HeJ (haplotype k) x BALB/c (haplotype d). In our assay, we find that the frequency for conversion of this particular DNA segment is 2 x 10(-6). This frequency is relevant only in the germ line; when liver cells were tested as an exampl...
Fertility and Sterility, 2010
Objective: To report a more quantitative approach to study the influence of varying levels of sperm DNA damage on transgenerational changes in genomic instability in a mouse model. Design: Experimental prospective study. Setting: Embryology research laboratory. Animal(s): Swiss albino mice. Intervention(s): The sperm DNA damage was induced by different doses of g-irradiation to male mice followed by mating with healthy female mice. Main Outcome Measure(s): Genomic integrity in embryos, fetus, and spermatozoa of F1 mice derived from the DNA-damaged sperm. Result(s): The transgenerational changes in genetic integrity were attributed by a dose-dependent increase in the frequency of micronuclei in preimplantation embryos and a concomitant increase in genomic instability in fetal liver cells and sperm chromatin modifications in F1 males. A strong positive correlation was observed between the extent of sperm DNA damage and somatic and germ-line genomic instability. Conclusion(s): Sperm-mediated transgenerational genomic instability is dependent on the amount of DNA damage present in the sire's sperm at the time of fertilization. (Fertil Steril Ò 2010;93:2486-90. Ó2010 by American Society for Reproductive Medicine.
Origins and consequences of DNA damage in male germ cells
Reproductive BioMedicine Online, 2007
He moved to the University of Newcastle in 1998 from the University of Edinburgh, UK, where he held an Honorary Professorship in the Faculty of Medicine and a special appointment with the MRC Reproductive Biology Unit. His research interests are focused on the differentiation of male and female germ cells and the practical application of that knowledge in a clinical and biotechnology context.