Mutagenesis vol. 18 no. 4 pp. 365-370,
July 2003
© 2003 UK Environmental Mutagen Society/Oxford University Press
Pms2 deficiency results in increased mutation in the Hprt gene but not the Tk gene of Tk+/– transgenic mice
Division of Genetic and Reproductive Toxicology, HFT-120, National Center for Toxicological Research, 3900 NCTR Road, Jefferson, AR 72079, USA
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The effects of deficiency in the DNA mismatch repair (MMR) protein Pms2 were investigated using the endogenous mouse Hprt and Tk genes as reporters of intragenic mutation and loss of heterozygosity (LOH). Pms2–/–Tk+/–, Pms2+/+Tk+/–, Pms2+/–Tk+/– and Pms2–/–Tk–/– mice were bred from Pms2+/–Tk+/– mice. At 2 months of age, the body weight and splenic T lymphocyte yields were significantly lower in Pms2–/–Tk–/– mice than in littermates of the other genotypes. The mice were evaluated for their spontaneous mutant frequencies in the Hprt and Tk genes of splenic lymphocytes and their frequency of micronuclei in polychromatic erythrocytes from bone marrow. The cloning efficiency of lymphocytes derived from Pms2–/–Tk–/– animals was 12-fold lower than that of animals of the other genotypes. Compared with Pms2+/+ and Pms2+/– mice, Pms2–/– mice had a 21- to 69-fold increase in the Hprt mutant frequency. The Hprt mutant frequency was equally high in Pms2-deficient, Tk+/– and Tk–/– mice. No significant Pms2-dependent change in mutant frequency was detected using the Tk mutational target. When individual Tk mutants were analyzed for LOH mutation by allele-specific genotyping, the fraction of LOH mutants was lower in Pms2-deficient than in Pms2-proficient mice (29.2 and 43.6%, respectively). The frequency of bone marrow micronuclei was significantly higher in Pms2–/–Tk–/– mice than in Pms2–/–Tk+/– mice. These observations suggest that the simultaneous occurrence of Tk and Pms2 deficiencies may cause a decrease in cell viability that diminishes the Tk mutational response, making it impossible to discern clearly the effect of Pms2 deficiency on LOH-type mutation using the Tk reporter system. The interaction between Tk deficiency and a component of the MMR system suggests that Tk-deficient cells may have higher levels of DNA polymerase misincorporation or endogenous DNA damage than Tk-proficient cells.
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Introduction |
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Pms2 is a component of the mouse DNA mismatch repair (MMR) system. Mouse MutL
is a heterodimer composed of the Mlh1 gene product and the Pms2 gene product. MutL is considered the major MutL species involved in murine MMR but other MutL species are known; MutLß is composed of Mlh1 and Pms1, and Mlh3 may also interact with Mlh1 (Prolla et al., 1998
). MutL heterodimers function in concert with a MutS heterodimer (i.e. MutS composed of Msh2 and Msh6 or MutSß composed of Msh2 and Msh3).
While the MutS heterodimers directly bind DNA mispairs, the function of the MutL heterodimer in less clear. Because MutL proteins are thought to coordinate protein–protein interactions, they have been called ‘molecular matchmakers’ (Sancar and Hearst, 1993). The N-terminal regions of MutL family members have been conserved and possess both ATPase and DNA-binding activities (Guarne et al., 2001). Female Pms2-deficient transgenic mice are fertile but Pms2-deficient males are sterile due to abnormalities in chromosome synapsis of meiotic prophase I (Baker et al., 1995).
The components of MMR contribute to four distinct cellular functions: (i) repair of DNA replication errors; (ii) response to DNA damage; (iii) inhibition of recombination between non-identical DNA sequences; and (iv) repair of mismatches in recombinational intermediates (Harfe and Jinks-Robertson, 2000). The loss of Pms2 function results in an 
100-fold increase in the spontaneous mutant frequency in a number of different tissues of Pms2-deficient mice as measured using a SupF transgenic reporter gene (Narayanan et al., 1997). When cII and lacI were used as mutational targets, 5- and 10-fold increases in spontaneous mutant frequency were detected, respectively (Andrew et al., 2000). A 13-fold increase in spontaneous lacI mutant frequency was measured in the epithelial cells of the small intestine of mice deficient in Pms2 (Baross-Francis et al., 2001). Mutant frequencies in the Aprt and Hprt genes of splenic T lymphocytes from Pms2-deficient mice were 3- and 48-fold higher than those of Pms2-proficient mice, respectively (Shao et al., 2002). The spontaneous mutational spectra of Pms2 mice are dominated by transitions and +/–1 frameshifts. Furthermore, direct PCR analyses of mononucleotide repeat tracts showed that such tracts were much more unstable in Pms2-deficient mice than in wild-type mice (Yao et al., 1999). This microsatellite instability (MSI) has also been detected in Pms2-deficient spermatocytes (Prolla et al., 1998)
MMR proteins function in the response to DNA damage. Generally, MMR-proficient cells are sensitive to the cytotoxic effects of DNA damaging agents while MMR-deficient cells are resistant (Wu et al., 1999). Direct interactions occur between MutS and the damaged DNA bases induced by O6-methylguanine, cisplatin, UV photoproducts, 2-aminofluorene, N-acetyl-2-aminofluorene and benzo[a]pyrene diol epoxide (Wu et al., 1999). Cell cycle arrest is normally triggered in response to DNA damage. Deficiency in MMR, including Pms2 deficiency, results in an inability to generate this response (Zeng et al., 2000). Pms2-deficient mice demonstrate alkylation tolerance (Qin et al., 1999, 2000).
There are two known MMR functions that involve recombination, repair of mismatches in recombinational intermediates and inhibition of recombination between non-identical DNA sequences (anti-recombination). Both of these functions have been primarily studied in yeast, with relatively little information available from mammalian systems. Anti-recombination blocks meiotic and mitotic recombination between divergent DNA sequences. The yeast Pms2 homolog, pms1, is required for the anti-recombination function observed in meiotic cells (Hunter et al., 1996). Evidence from cultured mouse embryonic stem cells, however, suggests that Msh2 and Msh6 but not Pms2 function is required for anti-recombination. Furthermore, a double-strand break-induced increase in crossing-over between nearly identical sequences was detected in Msh2 and Msh6 mutant cells, but not in Pms2 mutant cells (Inbar et al., 2000).
The role of MMR proteins in repairing mismatches in recombinational intermediates has also been well documented in yeast, where recombination leads to heteroduplex tracts. Repair of heteroduplex tracts is detected as gene conversion and the lack of repair is detected as post-meiotic segregation (PMS) (Alani et al., 1994). In general, MMR deficiency in yeast results in an increase in PMS (Borts et al., 2000). While 46–73% of the non-Mendelian segregations observed in yeast strains deficient in pms1 or msh2 were PMS events, 100% of the non-Mendelian segregations produced in a pms1/msh2 double-deficient strain were due to PMS (Alani et al., 1994). It has also been shown that in yeast msh4, msh5, mlh1 and mlh3 are directly involved in promoting crossing-over during meiosis (Harfe and Jinks-Robertson, 2000). Two types of data indicate that Mlh1 is needed for meiotic crossing-over in the mouse (Baker et al., 1996). First, the Mlh1 protein was localized to recombination nodules in Mlh1-proficient mice and, second, chiasmata were absent in Mlh1-deficient mice. Evidence of a direct role for Pms2 in meiotic recombination has not been reported. Currently, the only evidence suggesting that Pms2 may play a role in mouse meiotic recombination is the defect in chromosome pairing observed during spermatogenesis, a phenotype known to reflect decreased recombination.
The paucity of information on the effects of MMR deficiency on recombination in mitotic mammalian cells reflects, in large part, the dearth of useful assays for measuring this endpoint. Currently, there are two transgenic mouse models that can be used to detect such events, mice in which one copy of either the Aprt or the Tk gene has been inactivated by insertion of a neo cassette (Stambrook et al., 1996; Wijnhoven et al., 1998; Dobrovolsky et al., 1999). Using these models, mutants that have lost the function of the remaining normal copy of the reporter gene can be cultured, quantified and the fraction of the mutants produced by loss of heterozygosity (LOH) subsequently identified by PCR. In the Aprt transgenic mouse model, Pms2 deficiency caused an increase in recoverable intragenic mutations but had no apparent affect on LOH mutant frequency (Shao et al., 2002). In order to investigate whether deficiency in Pms2 affects the frequency of LOH in the Tk gene, we produced Pms2-deficient Tk+/– mice, determined the levels of Hprt and Tk mutant splenic T lymphocytes in these mice and determined the frequency of LOH-type mutation among the Tk mutant lymphocytes. Because a recombination defect might result in an increase in the formation of micronuclei, levels of bone marrow micronuclei were also determined for Pms2 and/or Tk mutant mice.
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Mice
Animal procedures were approved by the NCTR Institutional Animal Care and Use Committee. Pms2+/– mice were kindly provided by Dr Sean Baker (University of California, Berkeley, CA). Pms2+/– mice were bred with Tk+/– mice to produce Pms2+/–Tk+/– offspring. Pms2+/–Tk+/– mice were interbred to produce Pms2+/+Tk+/–, Pms2+/–Tk+/–, Pms2–/–Tk+/– and Pms2–/–Tk–/– mice. The Tk genotype of each mouse was determined using allele-specific PCR with primers TK14, TK16 and NEO4 as previously described (Dobrovolsky et al., 2000, 2003). The Pms2 genotype was determined using primers P2-1, P2-3 and NEO4, which generate an
300 bp product from the normal Pms2 allele and an 900 bp product from the targeted allele (Baker et al., 1995; Dobrovolsky et al., 2000). Each 25 µl Pms2 genotyping reaction contained 5 µl tail DNA (prepared using a DNeasy tissue kit; Qiagen, Valencia, CA), 200 nM each primer, 200 µM each dNTP, 1x HotStarTaq buffer and 2 U of HotStarTaq DNA polymerase (Qiagen). The cycling conditions were 15 min at 95°C, followed by 1 min at 94°C, 1 min at 58°C and 2 min at 72°C for 30 cycles, followed by 7 min at 72°C.
Hprt and Tk mutant lymphocytes
Animals were killed at 2 months of age. Splenic T lymphocytes were isolated by density gradient centrifugation as described (Meng et al., 1998). Lymphocytes were stimulated with a mitogen, diluted and plated using three 96-well microtiter plates at a concentration of 40 000 lymphocytes/well in the presence of 6-thioguanine for selection of Hprt mutants or using five 96-well plates at 10 000 lymphocytes/well in the presence of 5-bromodeoxyuridine for selection of Tk mutants. Clones resistant to 6-thioguanine or 5-bromodeoxyuridine were determined with an automated counting procedure using a fluorescent viability indicator (Dobrovolsky et al., 2000). Some lymphocytes were plated using two 96-well plates at 8 lymphocytes/well in the absence of selection to determine the cloning efficiency (CE) of each lymphocyte population. Mutant frequencies were calculated using the Poisson distribution (Jones et al., 1985). LOH-type Tk mutations in 5-bromodeoxyuridine-resistant clones were identified by allele-specific PCR using TK14, TK16 and NEO4 as previously described (Dobrovolsky et al., 1999).
Micronucleus detection
Bone marrow samples were flushed from the femur with 1 ml of phosphate-buffered saline (PBS) containing 10% fetal bovine serum (FBS) (Hyclone, Logan, UT) using a 3 ml syringe fitted with a 25 gauge needle. The samples were collected in microfuge tubes and then centrifuged at 8000 r.p.m. for 5 min. The supernatants were removed and the pellets triturated with 700 µl of PBS/10% FBS. Microscope slides were smeared with 10–20 µl of each cell suspension and air dried overnight. The slides were then fixed for at least 15 min with 100% methanol and air dried. After drying, the slides were stained for 1 min with a solution of 12.5 mg acridine orange (Sigma, St Louis, MO) in 100 ml of Sörenson’s buffer (Sigma) (Tinwell and Ashby, 1989). The slides were rinsed twice, for 10 and 15 min in Sörenson’s buffer. The slides were air dried and then stored in the dark at 4°C. Coverslips were mounted using 10–20 µl of Sörenson’s buffer immediately prior to scoring using a fluorescent microscope. At least 1000 polychromatic erythrocytes (PCEs) were scored per animal.
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Results |
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To analyze the effects of Pms2 deficiency on the mutational response at the Tk gene, Tk+/– mice with different Pms2 genotypes were needed. Therefore, Pms2+/–TK+/– mice were interbred and Tk+/– offspring with each of the three possible Pms2 genotypes (+/+, +/– and –/–) were collected. Pms2–/–Tk–/– mice were also collected so that the phenotype of double knockout mice could be examined. The double knockout animals (Pms2–/–Tk–/–) of both sexes were obviously smaller than their littermates of other genotypes. The weights of individual 2-month-old animals are given in Table I. The mean body weight of Pms2–/–Tk–/– males was 19.0 g, as compared with 23.4, 23.1 and 23.6 g for the Pms2+/+Tk+/–, Pms2+/–Tk+/– and Pms2–/–Tk+/– males, respectively. Similarly, the mean body weight of Pms2–/–Tk–/– females was 14.3 g, as compared with 19.2, 19.9 and 19.2 g for the Pms2+/+Tk+/–, Pms2+/–Tk+/– and Pms2–/–Tk+/– females, respectively. The lower body weights of the Pms2–/–Tk–/– animals were statistically significant for both sexes (P = 0.002, Bonferroni t-test). The spleens from the Pms2–/–Tk–/– animals appeared smaller and yielded significantly lower numbers of T lymphocytes than the spleens of other genotypes (P < 0.001, Bonferroni t-test). While an average of 12.9 x 106 lymphocytes were isolated from the Pms2–/–Tk–/– mice, an average of 37.9 x 106, 32.0 x 106 and 44.0 x 106 lymphocytes were isolated from the spleens of Pms2+/+Tk+/–, Pms2+/–Tk+/– and Pms2–/–Tk+/– mice.
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To determine the Hprt and Tk mutant frequencies in these lymphocyte populations, lymphocytes were cultured in the presence of 6-thioguanine or 5-bromodeoxyuridine, the mutant selection conditions for the Hprt and Tk assays, respectively. Additional lymphocytes were cultured in the absence of selection to determine the CE of each lymphocyte population. The CE and Hprt and Tk mutant frequencies determined for each animal are given in Table I, as well as the average CE and average Hprt and Tk mutant frequencies determined for each genotype. The mean CE of the Pms2–/–Tk–/– lymphocytes (1.19%) was significantly lower than that measured for the lymphocytes of all other genotypes (P < 0.001, Bonferroni t-test).
The spontaneous Hprt mutant frequencies measured in the Pms2+/+ and Pms2+/– animals were low, 0.4 and 1.3 x 10–6, respectively. The spontaneous Hprt mutant frequency was considerably higher in the Pms2–/– animals, 27.7 and 23.9 x 10–6 for the Tk+/– and Tk–/– genotypes, respectively. In contrast, the spontaneous Tk mutant frequency was approximately the same for each of the three Pms2 genotypes (30.2, 31.6 and 25.3 x 10–6 for the Pms2+/+, Pms2+/– and Pms2–/– genotypes, respectively).
The Tk mutant clones were characterized using allele-specific PCR. If both the targeted (neo gene insert) and wild-type Tk alleles were detected by PCR, then the clone was considered to have an intragenic (point) mutation. If the targeted allele produced a predominant PCR product, the mutant was considered to have an intergenic LOH mutation. The percentage of LOH mutation detected within the pool of mutant clones from each animal is given in Table II. The median frequency of LOH mutation measured in Tk mutant lymphocytes from Pms2–/– animals was 29.2%. A somewhat higher average LOH frequency (43.6%) was measured in the mutant lymphocytes from Pms2+/+ animals (P = 0.072, Mann–Whitney rank sum test comparing LOH in Pms2–/– and Pms2+/+ mice). The absolute frequency of intragenic mutation was virtually identical in Pms2-proficient and Pms2-deficient animals (17.0 x 10–6 versus 17.9 x 10–6, respectively). However, the absolute frequency of LOH-type mutation in the Pms2-proficient animals was almost twice that of the Pms2-deficient mice (13.2 x 10–6 versus 7.4 x 10–6).
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We hypothesized that if Pms2 is needed to successfully complete recombination, then bone marrow PCEs of Pms2-deficient mice might have an increased spontaneous micronucleus frequency. Therefore, bone marrow PCEs were isolated from femurs of Pms2+/+Tk+/–, Pms2+/–Tk+/+, Pms2+/+ Tk–/–, Pms2–/–Tk+/– and Pms2–/–Tk–/– mice. The data from three Pms2+/+Tk+/– and two Pms2+/–Tk+/+ animals were pooled because all of these animals are Pms2- and Tk-proficient and had similar micronucleus frequencies. The average micronucleus frequency in these control animals was 0.53 ± 0.12% (Table III). When the micronucleus frequencies from Pms2 knockout, Tk knockout and double knockout animals were compared with these controls, the double knockout mice (deficient in both Pms2 and Tk) had a micronucleus frequency that was significantly higher than that of all other genotypes (1.35 ± 0.25%, Bonferroni t-test, P < 0.05). While the average micronucleus frequency of Pms2+/+Tk–/– mice (0.87 ± 0.27%) was somewhat higher than the control animals, the difference was not statistically significant (Bonferroni t-test, P = 0.44).
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Discussion |
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The induction of spontaneous intragenic mutation that results from Pms2 deficiency is well documented but the potential role of Pms2 in intergenic mutation is relatively unexplored. Therefore, the effect of Pms2 deficiency on the frequency of LOH-type mutation was investigated using mice heterozygous for the Tk reporter gene, one of the few in vivo reporter systems that can be used to measure this type of mutation. It was expected that one of the three Tk mutational responses shown in Figure 1A would be observed. The frequency of intragenic mutation was expected to increase in Pms2–/– mice because a spontaneous induction of intragenic mutation (mainly point mutations and frameshifts) had been reported for each mutational target that has been analyzed on a Pms2–/– background (Narayanan et al., 1997
; Andrew et al., 2000; Baross-Francis et al., 2001; Shao et al., 2002). What was uncertain was whether the absolute frequency of LOH mutation would decrease, remain unchanged or increase. Given the interrupted Tk homology that results from neo insertion in the targeted Tk allele, an increase in LOH might be expected due to a loss of an anti-recombination activity in Pms2–/– mice. Conversely, if Pms2 has a function in promoting homologous recombination or in recombinational repair, then the frequency of LOH would be expected to decrease in the Pms2–/– mice. Surprisingly, the overall Tk mutant frequency in the Pms2-deficient animals decreased slightly (30.2 x 10–6 to 25.3 x 10–6) and the absolute frequency of intragenic mutation in Tk was virtually unchanged (17.0 x 10–6 and 17.9 x 10–6 in Pms2+/+ and Pms2–/– mice, respectively) (Figure 1B). This contrasts sharply with the Hprt mutational response where an 60-fold induction of Hprt mutation was detected in Pms2–/– animals. This Hprt induction in Pms2-deficient animals is consistent with that measured in the Aprt transgenic mouse (Shao et al., 2002).
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From this study it appears that the Pms2–/–Tk–/– genotype impacts cell growth and viability. The Pms2–/–Tk–/– mice were smaller, their spleens were smaller and yielded fewer T lymphocytes and the CE of these splenic lymphocytes was considerably lower than that of all other genotypes. In terms of the relatively low lymphocyte CE and yield, the Pms2–/–Tk–/– mice are similar to mice deficient in the Tk gene alone (Dobrovolsky et al., 2003
). However, the significant growth retardation evident by 2 months of age in the Pms2–/–Tk–/– mice is a feature of only the double knockout mice. In addition, a statistically significant increase in micronucleus frequency was seen in the bone marrow PCEs of the double knockout mice, suggesting an increased sensitivity to spontaneous mutational processes. Furthermore, an interaction between Pms2 and Tk deficiencies that reduced cell viability would explain why spontaneous Hprt mutation but not Tk mutation was increased in the Pms2-deficient cells. A mutant frequency is determined by dividing the number of mutant clones that grow in the selective medium by the number of clonable cells as measured on CE plates without selective medium. For the Hprt calculations, both the selected and unselected cells have the same Pms2 and Tk genotypes so the same plating efficiency is expected on the CE and Hprt mutant selection plates. In Pms2–/–Tk–/– mice, for instance, the same (low) CE should occur on both the CE and Tk mutant selection plates so a decrease in Pms2–/–Tk–/– cell viability would not affect the calculation of Hprt mutant frequency. In the Tk assay of Pms2–/–Tk+/– mice, however, the population of Tk mutants (Pms2–/–Tk–/–) may have a lower CE (like the 1.19% measured in double knockout mice; Table I) than the population of mostly Pms2–/–Tk+/– cells plated to measure CE (14.3%, Table I). Consequently, the apparent 12-fold decrease in Pms2–/–Tk–/– cell viability may cause the calculated Tk mutant frequency of the Pms2–/–Tk+/– mice to be an underestimate of the actual spontaneous mutant frequency. The absolute frequency of intragenic mutation calculated for Pms2–/–Tk+/– and Pms2+/+Tk+/– mice was nearly identical (Table I and Figure 1), but the level in Pms2–/–Tk+/– mice would be 12-fold higher if a correction was made for the effect of CE. In other mutational assays, the Pms2–/– genotype has caused a 3- to 100-fold induction of mutation. An almost 2-fold decrease in the absolute frequency of LOH mutants was observed in the Pms2–/–Tk–/– mice but, given the decreased viability of the Pms2- and Tk-deficient lymphocytes, it is difficult to draw any conclusions regarding the impact of Pms2 deficiency on the recombinational processes involved in LOH.
It is unclear why Pms2–/–Tk–/– cells have decreased viability. Because Tk is an enzyme involved in the salvage pathway of nucleotide synthesis, it seems most probable that Tk–/– animals may have aberrant nucleotide pool levels that could result in increased nucleotide misincorporation by DNA polymerase. The measurement of increased thymidine in the serum of Tk–/– mice supports the idea that these animals may have unbalanced nucleotide pools (Dobrovolsky et al., 2003). If the DNA of double knockout cells has higher levels of nucleotide misincorporation, this could subsequently interfere with the repair of other types of endogenous DNA damage.
Nucleotide misincorporation is normally corrected by MMR, but the Pms2–/– mice lack this function. Therefore, polymerase misinsertion and endogenous DNA damage must be corrected using some other type of repair. Because Pms2 deficiency results in an inability to initate cell cycle arrest in response to DNA damage, damaged cells might continue to divide and accumulate damage until they are no longer viable. Other repair processes may also be compromised by Pms2 deficiency. Another possibility is that recombinational repair is initiated in Pms2-deficient cells but not successfully completed in some proportion of them. The increased micronucleus frequency in bone marrow PCEs of Pms2–/– mice might be an indication of such unproductive recombinational repair.
This study provides new phenotypic information for the various Pms2 and Tk genotypes, which is summarized in Figure 2. However, this study also highlights some of the difficulties in investigating the potential role of MMR proteins in mammalian mitotic recombination. The apparent redundancy and versatility of MMR components, as well as interactions between various repair processes, makes it difficult to clearly define MMR functions, even using transgenic technology. Also, the recently discovered chromosomal proximity of the kynurenine formamidase (KF) gene to the Tk gene suggests that Tk knockout animals may also be deficient for the KF gene (Schuettengruber et al., 2003). Thus, a potential complication in interpreting the mutational data from Pms2–/–Tk+/– mice is the possibility that Tk LOH mutants might actually have a triple knockout genotype. Nevertheless, this study provides new insights regarding the cellular consequences of Tk deficiency and its potential interaction with a deficiency in MMR.
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Acknowledgements |
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We thank Dr Sean Baker (University of California, Berkeley, CA) for providing the Pms2 mice. We thank the NCTR Animal Care and Veterinary Services staff for their assistance in establishing and maintaining the Pms2 and Tk transgenic mouse colonies.
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Notes |
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1To whom correspondence should be addressed. Tel: +1 870 543 7946; Fax: +1 870 543 7393; Email: bparsons{at}nctr.fda.gov
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References |
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Received on December 23, 2002; accepted on March 26, 2003.
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