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Cancer Research 68, 3630, May 15, 2008. doi: 10.1158/0008-5472.CAN-08-0484
© 2008 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

Single-Molecule PCR Analysis of Germ Line Mutation Induction by Anticancer Drugs in Mice

Colin D. Glen1, Andrew G. Smith2 and Yuri E. Dubrova1

1 Department of Genetics and 2 Medical Research Council Toxicology Unit, University of Leicester, Leicester, United Kingdom

Requests for reprints: Yuri Dubrova, Department of Genetics, University of Leicester, University Road, Leicester LE1 7RH, United Kingdom. Phone: 44-116-252-5654; Fax: 44-116-252-3378; E-mail: yed2{at}le.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Understanding and estimating the genetic hazards of exposure to chemical mutagens and anticancer drugs in humans requires the development of efficient systems for monitoring germ line mutation. The suitability of a single-molecule PCR–based approach for monitoring mutation induction at the mouse expanded simple tandem repeat (ESTR) locus Ms6-hm by chemical mutagens and anticancer drugs has been validated. The frequency of ESTR mutation was evaluated in the germ line of male mice exposed to the well-characterized alkylating agent and mutagen, ethylnitrosourea, and four widely used anticancer drugs, bleomycin, cyclophosphamide, mitomycin C, and procarbazine. The dose-response of ethylnitrosourea-induced mutation was found to be very close to that previously established using a pedigree-based approach for ESTR mutation detection. Paternal exposure to the clinically relevant doses of bleomycin (15–30 mg/kg), cyclophosphamide (40–80 mg/kg), and mitomycin C (2.5–5 mg/kg) led to statistically significant, dose-dependent increases in ESTR mutation frequencies in the germ line of treated male mice. Exposure to procarbazine led to a maximal increase in mutation frequency at 50 mg/kg, with a plateau at the higher concentrations. The results of this study show that the single-molecule PCR technique provides a new and efficient experimental system for monitoring the genetic effects of anticancer drugs, capable of detecting increases in mutation rates at clinically relevant doses of exposure. In addition, this approach dramatically reduces the number of mice needed for the measurement of germ line mutation induction. [Cancer Res 2008;68(10):3630–6]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
In recent decades, improvements in cancer treatment have produced dramatic increases in survival rates among patients. For example, in the United States, between 1985 and 1999, the 5-year survival rate for children diagnosed with cancer was 75.8% (1). Furthermore, the prevalence of childhood cancer survivors among young American adults ages 20 to 39 is approximately 1 in 640 (2). However, along with surgery, the mainstays of cancer treatment are radiotherapy and chemotherapy, both of which are genotoxic and mutagenic. The survivors of therapy can experience the late adverse effects, including the development of secondary, treatment-related tumors (3). Another major adverse effect of radiotherapy and chemotherapy is mutagenesis, which can cause heritable genetic damage and diseases in the offspring of survivors (4). It should be stressed that the majority of chemotherapy treatments compromise normal sexual function and often lead to prolonged infertility (5). The long-term genetic effects of parental exposure to anticancer drugs therefore represent a major concern to genetic toxicology.

The analysis of germ line mutation induction in mice currently provides the main source of experimental data for evaluating the genetic risk of exposure to chemical mutagens and anticancer drugs in humans (4, 6). The majority of the mouse data have been generated using either the specific locus method (Russell 7-locus test) or the dominant lethal test (4, 6). Both tests provide important information on the mutagenicity of chemicals and the stage-specificity of mutation induction, but require the profiling of very large numbers of exposed mice and their offspring, which increasingly, is unacceptable in terms of animal usage. Most importantly, these tests lack the sensitivity to detect mutation induction in the germ line of mice exposed to low and often intermediate concentrations of chemical mutagens. Given that these are the doses of most concern to genetic toxicology, the development of new, more sensitive and efficient approaches for evaluating the germ line effects of chemical mutagens is warranted.

We have previously developed a highly sensitive technique for monitoring radiation-induced mutation in the germ line of male mice (7–9). This technique employs highly unstable expanded simple tandem repeat (ESTR) loci. These loci were originally termed minisatellites, but have been renamed to distinguish them from the much more stable true minisatellites in the mouse genome (10). Unstable ESTR loci consist of homogenous arrays of short repeats (4–6 bp) and show very high spontaneous mutation rates in the mouse germ line, observed as size changes in the alleles of these loci (10–12). The analysis of ESTR mutation induction in the germ line of male mice has shown that exposure to ionizing radiation results in elevated mutation rates, detectable at doses that were previously inaccessible using standard approaches for monitoring germ line mutation in mice (7–9, 13). We have also conducted a detailed analysis of ESTR mutation induction in the germ line of male mice exposed to some well-recognized mutagens and anticancer drugs (13, 14). The results of these studies were consistent with those obtained earlier using traditional approaches to assess germ line mutation in mice, thus further validating the ability of ESTR loci to reflect DNA damage in the mouse germ line. It should be stressed that the results of our studies also show the high efficiency of ESTR mutation induction after acute exposure of the early premeiotic stages of mouse spermatogenesis (8, 13, 14); thus, making these loci a useful tool for the analysis of long-term genetic effects of paternal exposure, similar to that following cancer chemotherapy.

It should be noted that the abovementioned ESTR data were obtained using a pedigree-based approach which requires the profiling of several hundred offspring of exposed parents to detect significant changes. Although this compares favorably with the specific locus method, in which analysis of thousands or even hundreds of thousands of mice is required, it has been found that a single-molecule PCR (SM-PCR)–based technique for monitoring ESTR mutation can be used to further reduce the number of mice needed to measure radiation-induced changes in germ line mutation frequencies (15). This approach involves diluting bulk sperm genomic DNA and amplifying multiple samples of DNA, each containing approximately one ESTR molecule, and allows the detection of a indefinitely large number of de novo mutants in a single male. Here, we present the results of the first extensive study aimed at validating the suitability of the SM-PCR technique for monitoring mutation induction in the germ line of male mice exposed to chemical mutagens and anticancer drugs. We have also investigated the sensitivity of this technique for the analysis of germ line mutation induction within a clinically relevant dose range of exposure to anticancer drugs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Animals. Male 6-week-old C57BL/6 x CBA/Ca F1 hybrid mice were obtained from Harlan. Animals received a single dose of the test compound, dissolved in PBS and delivered via i.p. injection, at 8 weeks old. Caudal epididimy were collected 8 weeks after treatment and from age-matched 16-week-old untreated control mice. Animal procedures were carried out under Home Office project license no. PPL 40/2605.

Chemicals. Bleomycin sulfate, cyclophosphamide, ethylnitrosourea, and mitomycin C were obtained from Sigma-Aldrich. Procarbazine was obtained from Sequoia Research Products, Ltd.

Preparation of sperm DNA. Sperm DNA samples were prepared in a laminar flow hood as previously described (15, 16). Approximately 5 µg of each DNA sample was digested with 20 units of MseI for 2 h at 37°C; MseI cleaves outside the ESTR array and distal to the PCR primer sites used for PCR amplification and was used to render genomic DNA fully soluble prior to dilution. Each digested DNA sample was diluted to ~10 ng/mL in 5 mmol/L of Tris-HCl (pH 7.5) in the presence of 5 µg/mL of carrier herring sperm DNA (Sigma) prior to mutation analysis.

Mutation detection. The frequency of ESTR mutation was evaluated using SM-PCR (15, 16). The Ms6-hm ESTR locus was amplified in 10 µL reactions using 0.4 µmol/L flanking primers HM1.1F (5'-AGA GTT TCT AGT TGC TGT GA-3') and HM1.1R (5'-GAG AGT CAG TTC TAA GGC AT-3'). The Expand High-fidelity PCR System (Roche) was used (0.035 units/µL) with 1 mol/L of betaine and 200 µmol/L each of deoxynucleotide triphosphate. Amplification was carried out at 96°C (20 s), 58°C (30 s), and 68°C (8 min) for 30 cycles on a PTC-225 DNA Engine Tetrad (MJ Research). To increase the robustness of the estimates of individual ESTR mutation frequencies, on average, 135 amplifiable molecules were analyzed for each male mouse.

PCR products were resolved on a 40-cm-long agarose gel and detected by Southern blot hybridization as previously described (8). Following Southern blot hybridization, autoradiographs were scored by two independent observers. The frequencies of ESTR mutation and standard errors were estimated using a modified approach proposed by Chakraborty (17). DNA fragment sizes were estimated by the method of Southern (18), with a 200-bp DNA Step Ladder (Promega) included on all gels.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
Experimental design. Mutations were scored at both the smaller (~3 kb) CBA/Ca-derived allele and the larger (4–5 kb) C57BL/6 allele. As in our previous studies, only bands showing a shift of at least 1 mm relative to the progenitor allele were scored as mutants (Fig. 1 ).


Figure 1
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  		Figure 1. Mutation detection at the Ms6-hm locus by SM-PCR in C57BL/6 x CBA/Ca F1 hybrid mice. Arrows, mutations at the CBA/Ca (A) and C57BL/6 (B) alleles. In both examples, repeat units were lost.

 
The well-characterized alkylating agent and mutagen, ethylnitrosourea, was used as a test compound to validate the SM-PCR technique. Mutation induction by four diverse anticancer drugs, bleomycin, cyclophosphamide, mitomycin C, and procarbazine, was then measured. The doses of exposure to anticancer drugs were calculated according to Food and Drug Administration guidelines recommending multiplying human doses by 12.3 per kilogram of bodyweight to give equivalent doses for mice (19). By sacrificing animals 8 weeks after treatment, it was ensured that the sperm assessed were derived from exposed As spermatogonia (20).

Ethylnitrosourea. Ethylnitrosourea is one of the best-characterized chemical mutagens in the mouse germ line (21). Using the pedigree-based approach, we have previously shown that premeiotic exposure to ethylnitrosourea doses of between 12.5 and 75 mg/kg produced highly significant increases in ESTR mutation rates (14). We found that similar increases could be detected using SM-PCR when measuring the effects of 12.5, 25, and 50 mg/kg of ethylnitrosourea. All three doses produced significant increases in mutation frequencies (Table 1 ). The pattern of ESTR mutation induction was linear within the interval of doses from 12.5 to 50 mg/kg (Fig. 2A ) and, most importantly, was very close to that previously established by conventional pedigree-based analysis (Fig. 2B). This apparent similarity clearly shows that SM-PCR can be used to reliably monitor mutation induction in the male germ line following exposure to chemical mutagens.


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  		Table 1. Summary of mutation data

 

Figure 2
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  		Figure 2. ESTR mutation frequencies in ethylnitrosourea-treated male mice, determined by SM-PCR (A) and from pedigree data (B). The pedigree data for ethylnitrosourea-exposed CBA/Ca males are taken from ref. 14. Points, mean; bars, SE.

 
Bleomycin. Bleomycin is widely used in the treatment of squamous cell cancers, melanoma, sarcoma, testicular cancer, and Hodgkin's and non–Hodgkin's lymphomas (22). Human single-dose exposure to this highly toxic antibiotic does not exceed 15 units/m2 (23); the equivalent mouse dose is between 7 and 8 mg/kg. As exposure to bleomycin mainly results in the induction of double-strand breaks, it has been classified as radiomimetic (22, 24). Previous analysis of mutation induction at specific loci has shown that its mutagenicity in male mice receiving a single dose of 30 mg/kg of bleomycin is restricted to premeiotic spermatogonia (25). It was also shown that paternal premeiotic exposure to smaller doses of this drug (<20 mg/kg) did not induce dominant-lethal mutations (25) or chromosome aberrations (26) in male mice. We found only a small, statistically nonsignificant increase in ESTR mutation frequency following exposure to 15 mg/kg of bleomycin, but a much more dramatic, significant increase after treatment with 30 mg/kg (see Table 1; Fig. 3A ). Our results are therefore in line with the specific-locus data, and together with the results previous studies (25, 26), also indicate that exposure to the clinically relevant doses of this anticancer drug (<10 mg/kg) may not result in a measurable mutation induction in the mouse male germ line. However, given that the mutagenicity of bleomycin in female mice substantially exceeds that for males (27), this notion should be treated with caution.


Figure 3
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  		Figure 3. ESTR mutation frequencies in DNA samples extracted from sperm of male mice exposed to the anticancer drugs bleomycin (A), cyclophosphamide (B), mitomycin C (C), and procarbazine (D). Points, mean; bars, SE.

 
Cyclophosphamide. Cyclophosphamide is a widely used anticancer drug and immunosuppressive agent which, following metabolic activation, forms DNA adducts, mostly N7-substituted guanine derivatives and phosphoester adducts (28, 29). Single doses for anticancer treatment in humans are typically between 400 and 1,200 mg/m2 (23); the equivalent mouse dose for this drug is 130 to 400 mg/kg. The stage-specificity of mutation induction by this drug is an issue of some controversy. Thus, the analysis of mutation induction at specific loci showed that paternal exposure to 120 mg/kg of cyclophosphamide mostly affects postmeiotic spermatids (30). However, according to the results of other studies, premeiotic exposure to this drug results in significant increases in the frequency of dominant mutations and chromosome aberrations in treated males (reviewed in ref. 28). We found that premeiotic spermatogonial exposure to cyclophosphamide resulted in a linear increase of ESTR mutation frequency in the germ line of male mice exposed to 40 and 80 mg/kg (see Table 1; Fig. 3B). In this respect, our results are at odds with the specific locus data showing that postmeiotic exposure to the high doses of this drug does not affect mutation rate in male mice (30). According to our previous results, the pattern of premeiotic mutation induction at ESTR and specific loci is similar (8, 13, 14), the cyclophosphamide data therefore indicate that premeiotic paternal exposure to this drug may be mutagenic. Given that highly significant increases in ESTR mutation rate were detected in male mice whose exposure was below a level equivalent to the doses typically administered to patients, our data raise the important issue of long-term genetic effects of paternal exposure to cyclophosphamide in humans.

Mitomycin C. A streptomycin-derived antibiotic, mitomycin C, is a widely used anticancer drug. Following metabolic activation, it forms a number of sequence-specific DNA adducts, including alkylated monoadducts and cross-linked adducts (31, 32). Human single doses are typically between 10 and 20 mg/m2 (23), with the equivalent mouse dose of 3 to 6 mg/kg. The results of previous studies have shown that exposure of male mice to >5 mg/kg of mitomycin C significantly increases the mutation rate in premeiotic spermatogonia (33, 34). Our SM-PCR data further show the mutagenicity of this drug. Doses of 2.5 and 5 mg/kg produced significant increases in ESTR mutation frequency that were dose-dependent (see Table 1; Fig. 3C). Taken together, these results suggest that exposure to the clinically relevant doses may be regarded as a potential long-term genetic risk factor for humans.

Procarbazine. Procarbazine is used to treat a number of cancers, including Hodgkin's lymphoma. Metabolites of this drug inhibit DNA polymerase and react directly with DNA (35). Human single doses do not exceed 150 mg/m2 (23); the equivalent mouse dose for this drug is ~50 mg/kg. The results of a previous study showed that the lowest dose of procarbazine that significantly increased mutation rate at the specific loci in male mice was 400 mg/kg (36). However, we found procarbazine to be maximally mutagenic in the germ line of male mice at 50 mg/kg (Table 1). The doubling of that dose produced no further increase in mutation frequency, suggesting that a plateau of ESTR mutation induction had been reached (Fig. 2D). We therefore suggest that exposure to the clinically relevant doses of this anticancer drug could have long-term mutagenic effects in the human germ line.

ESTR mutation spectrum. The incidence of ESTR mutations involving gain or loss was defined for 576 mutations found in control and exposed males. In all exposed groups, the incidence of ESTR mutations involving either the gain or loss of repeat units did not significantly differ from that in control males (Table 1). We next determined the spectra of ESTR mutations. This analysis was restricted by the resolution of agarose gel electrophoresis as the smallest mutational changes detectable at the CBA/Ca and C57BL/6 alleles were three and seven repeats, respectively (Fig. 1). Within each treatment group, the mutation spectra for the males exposed to different doses of mutagens did not significantly differ (data not shown). The combined distributions of length changes at ESTR loci were indistinguishable between controls and all exposed groups (Fig. 4 ). We therefore conclude that exposure to the anticancer drugs analyzed here does not affect the size of ESTR mutation changes. These data are in line with the results of our previous studies showing no difference in the structure of spontaneous and radiation- or ethylnitrosourea-induced ESTR mutants in the mouse germ line (37, 38). We and others have previously hypothesized that ESTR mutation induction cannot be attributed to the direct targeting of these small genomic loci by ionizing radiation and chemical mutagens (8, 9, 13, 14, 39). One of the arguments for nontargeted mechanisms comes from the comparison of ESTR mutation spectra in the germ line of control and exposed mice (37). Given the similarities in the structure of spontaneous and induced ESTR mutants reported here, our data indicate that mutation induction at ESTR loci by anticancer drugs may also result from the initial mutagen-related DNA damage elsewhere in the genome, and later, indirect mutation at these loci. The mechanisms underlying this nontargeted process are still unknown.


Figure 4
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  		Figure 4. Spectrum of germ line ESTR mutations in controls and exposed male mice (Kruskal-Wallis test, P = 0.7909). The progenitor allele was assumed to be the paternal allele closest in size to the mutant allele (see text for details).

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
The main purpose of our current study was to determine whether the SM-PCR approach could be used as a biomonitoring system to assess the germ line mutagenicity of anticancer drugs in mice. Thus far, the lack of sensitive and relatively inexpensive approaches has been a severe limitation to the in-depth study of germ line mutagenicity. Given that mutation induction in mice is still the main source of experimental data used to evaluate the genetic risk for humans, over recent years, a considerable effort has been made to develop more sensitive and efficient approaches for monitoring germ line mutation in mice. To date, a vast amount of experimental data has been obtained using two in vivo techniques—the transgenic mouse mutational assay (40) and pedigree-based ESTR mutation assay (41). In contrast with traditional germ line mutation assays, both techniques require the profiling of relatively small numbers of control and exposed mice. As far as the SM-PCR approach is concerned, the results of previous studies clearly show that even in comparison with our ESTR pedigree-based mutation assay, this technique dramatically reduces the number of mice needed for the measurement of germ line mutation frequencies in irradiated male mice (15, 42) and those exposed to environmental mutagens (43).

Given the results of our early studies demonstrating the high efficiency of pedigree-based ESTR mutation assay for the analysis of mutation induction in the germ line of male mice exposed to well-characterized chemical mutagens (14), we sought to extend this analysis to encompass a number of widely used anticancer drugs. As the efficiency of SM-PCR for monitoring germ line mutation in male mice exposed to chemical mutagens has never been properly validated, in this study, the pattern of ESTR mutation induction in the germ line of male mice exposed to the well-characterized mutagen, ethylnitrosourea, was first established. As the dose-response for ethylnitrosourea-induced mutation established by the SM-PCR technique was very close to that previously established by the conventional pedigree-based analysis (Fig. 2), we can therefore conclude that SM-PCR can be used to reliably monitor mutation induction in the male germ line following exposure to chemical mutagens.

We next addressed the important issue of sensitivity of SM-PCR for monitoring mutation induction in the germ line of male mice exposed to the four widely used anticancer drugs. Table 2 presents a comparison of the results on mutation induction in male mice following premeiotic exposure to ethylnitrosourea and anticancer drugs as obtained by the specific locus test (Russell 7-locus test) and SM-PCR. Apart from the obvious difference in the number of animals required for the analysis of mutation induction by the two techniques (850,860 versus 37), this comparison also revealed that the sensitivity of the SM-PCR technique exceeds that of the Russell 7-locus test. For all the anticancer drugs included in this study, we analyzed mutation induction within the range of the clinically relevant doses for humans, whereas many previous publications investigated the effects of exposure to much higher doses. The SM-PCR data presented here clearly show that the mutagenicity of some drugs, including, for example, cyclophosphamide, following premeiotic paternal exposure may be higher than previously reported, thus raising the important issue of their long-term genetic effects in humans. It should however be stressed that, although our results show the effects of premeiotic exposure to anticancer drugs, they do not provide enough evidence on whether the stability of stem cells in the germ line of treated animals may remain compromised over a substantial period of time. Future studies should address this important issue.


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  		Table 2. Comparison of the specific locus and SM-PCR methods for mutation detection

 
Recently, a number of transgenic mutational models were used to investigate germ line mutation induction in male mice exposed to ethylnitrosourea and the same anticancer drugs analyzed here. Although the number of animals required for the analysis of mutation induction by this technique and SM-PCR is similar, their sensitivities dramatically differ. For example, the MutaMouse transgenic model was only used for the analysis of mutation induction in the germ line of male mice exposed to 150 mg/kg of ethylnitrosourea (44–47), whereas in our current and previous studies (14), a significant increase in ESTR mutation frequency was detected at doses as low as 12.5 mg/kg. The same is also true for the anticancer drugs analyzed here. Thus, the analysis of premeiotic mutation induction in the germ line of BigBlue transgenic mice exposed to 100 mg/kg of cyclophosphamide yielded negative results (48), whereas our SM-PCR data clearly show high mutagenicity of this drug at doses as low as 40 mg/kg (Table 1). Premeiotic exposure to 4 mg/kg of mitomycin C did not affect the frequency of gpi-{delta} mutation in the germ line of transgenic male mice (40); in contrast, our data show that this anticancer drug is mutagenic at doses of ≤2.5 mg/kg. It was previously reported that doses of procarbazine as high as 750 mg/kg did not affect mutation rates at the lacZ gene in testicular DNA of MutaMouse transgenic males (49), yet in our study, a highly significant increase in ESTR mutation frequency was detected in the germ line of male mice exposed to just 40 mg/kg of procarbazine. Taken together, these data clearly show that the sensitivity of SM-PCR techniques to a wide range of chemical mutagens and anticancer drugs substantially exceeds that of a variety of mouse transgenic assays.

In summary, our results show that the SM-PCR technique offers a highly efficient and more ethically acceptable method of assessing the mutagenicity of anticancer drugs and other chemical mutagens. The main advantage of this assay is its ability to detect mutation induction in the germ line of male mice at doses that are in or even below the clinically relevant range. As ESTR mutation induction predominantly occurs at the early premeiotic stages of mouse spermatogenesis, the SM-PCR technique can therefore provide a useful tool for the analysis of long-term genetic effects of paternal exposure, which are similar to those after cancer chemotherapy. Given that this approach allows the recovery of large numbers of de novo mutants from single individuals, it dramatically reduces the number of mice needed for the measurement of germ line mutation induction. In addition, as SM-PCR directly establishes the frequency of ESTR mutation in the germ line of exposed parents, it also reduces experimental time by removing the necessity to breed them. Taken together, the results of this study show that the SM-PCR technique represents a very powerful and efficient tool for monitoring germ line mutation induction by a variety of mutagens, including commonly used anticancer drugs. It also seems that the ESTR assay should be included in a battery of existing tests for germ line mutagenicity. Given the unique mechanism(s) of spontaneous and induced ESTR mutation, we would recommend that this technique should be used in parallel with other traditional end points such as gene mutations, chromosome aberrations, etc.


   Disclosure of Potential Conflicts of Interest
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
Grant support: Medical Research Council grant G0300477/66802 (Y.E. Dubrova and A.G. Smith), and by the European Commission NOTE integrated project and Cancer Research UK C23912/A9483 (Y.E. Dubrova).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank the Division of Biomedical Services, University of Leicester (Leicester, UK) for their expert animal care and Dr. Ruth Barber for helpful discussion.

Received 2/ 7/08. Revised 3/12/08. Accepted 3/12/08.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

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R. J. Hardwick, M. V. Tretyakov, and Y. E. Dubrova
Age-Related Accumulation of Mutations Supports a Replication-Dependent Mechanism of Spontaneous Mutation at Tandem Repeat DNA Loci in Mice
Mol. Biol. Evol., November 1, 2009; 26(11): 2647 - 2654.
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