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Updated: Oct 1, 2026
Transgenic Rodent Assay for Quantifying Male Germ Cell Mutant Frequency
Published on: August 6, 2014
Detection of radiation and cyclophosphamide-induced mutations in individual mouse sperm at a human expanded
Yun Zhang1, Darren G Monckton, Michael J Siciliano
1Department of Experimental Radiation Oncology, The University of Texas M.D. Anderson Cancer Center, Houston 77030, USA. yzhang@ciit.org
Abstract:
A method to measure the germline mutations induced by cancer treatment in humans is needed. To establish such a method we used a transgenic mouse model consisting of a human DNA repeat locus that has a high spontaneous mutation frequency as a biomarker. Alterations in repeat number were measured in individual sperm from mice hemizygous for an expanded (CTG)(162) human myotonic dystrophy type 1 (DM1) microsatellite repeat using single genome-equivalent (g.e.) PCR and detection by a DNA fragment analyzer. Mutation frequencies were measured in DNA from sperm from controls and sperm derived from stem spermatogonia, differentiating spermatogonia, and spermatocytes exposed to radiation and from spermatocytes of mice treated with cyclophosphamide. There was no increase above control levels in mutations, scored as >1 repeat changes, in any of the treated groups. However, moderately large deletion mutants (between 9 and 20 repeat changes) were observed at frequencies of 2.2% when spermatocytes were treated with cyclophosphamide and, 1.8 and 2.5% when spermatocytes and stem cells, respectively, were treated with radiation, which were significantly higher than the frequency of 0.3% in controls. Thus, radiation and cyclophosphamide induced deletions in the expanded DM1 trinucleotide repeat. PCR artifacts were characterized in sperm DNA from controls and from mice treated with radiation; all artifacts involved losses of more than 20 DM1 repeats, and surprisingly the artifact frequency was higher in treated sperm than in control sperm. The radiation-induced increase in the frequency of PCR artifacts might reflect alterations in sperm DNA that destabilize the genome not only during PCR amplification but also during early embryonic development.
Insights
Cancer treatments like radiation and cyclophosphamide can induce germline mutations. This study used a mouse model to show these treatments cause DNA deletions in sperm, highlighting potential risks for future generations.
Area of Science:
- Genetics
- Molecular Biology
- Cancer Research
Background:
- A reliable method to detect germline mutations from cancer therapies in humans is crucial.
- Transgenic mouse models offer a valuable platform for studying mutation induction due to their controlled genetic makeup and high spontaneous mutation rates.
Purpose of the Study:
- To establish and validate a method for measuring germline mutations induced by cancer treatments.
- To assess the mutagenic effects of radiation and cyclophosphamide on sperm DNA using a human myotonic dystrophy type 1 (DM1) microsatellite repeat in mice.
Main Methods:
- Utilized a transgenic mouse model with an expanded human (CTG)(162) DM1 microsatellite repeat as a biomarker.
- Employed single genome-equivalent (g.e.) PCR and DNA fragment analysis to quantify repeat number alterations in individual sperm.
- Analyzed sperm DNA from control mice and those exposed to radiation or cyclophosphamide at various developmental stages (stem spermatogonia, differentiating spermatogonia, spermatocytes).
Main Results:
- No significant increase in mutations (>1 repeat change) was observed in most treated groups compared to controls.
- Cyclophosphamide treatment of spermatocytes and radiation treatment of spermatocytes and stem cells significantly increased moderately large deletions (9-20 repeats) in the DM1 locus.
- PCR artifacts, involving >20 repeat losses, were observed and surprisingly occurred more frequently in DNA from treated sperm, suggesting potential genome destabilization.
Conclusions:
- Radiation and cyclophosphamide can induce deletions in the expanded DM1 trinucleotide repeat in mouse sperm.
- The observed increase in PCR artifacts in treated sperm may indicate broader DNA instability with implications for early embryonic development.
- The developed method provides a sensitive approach to assess treatment-induced germline mutations, crucial for understanding long-term reproductive risks.
