Mutagenesis, Vol. 16, No. 1, 59-64,
January 2001
© 2001 UK Environmental Mutagen Society/Oxford University Press
The application of the restriction site mutation assay to compare 1-ethyl-1-nitrosourea-induced mutations between the endogenous p53 gene and the transgenic LacZ gene in MutaMouse testes
1 Institute of Biological Sciences, University of Aberystwyth, Aberystwyth, 2 Centre for Molecular Genetics and Toxicology, School of Biological Sciences, University of Wales Swansea, Singleton Park, Swansea and 3 Zeneca, Central Toxicology Laboratory, Alderley Park, Cheshire, UK
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Transgenic mouse modelling has provided a new approach to study the various steps involved in spontaneous and induced mutagenesis in rodent somatic and germline tissues in vivo. However, the important question arises as to whether mutations occur at the same rate in transgenes as in endogenous genes. Here, the restriction site mutation (RSM) assay was used to study mutations induced in the endogenous p53 gene and LacZ transgene of MutaMouse testes treated with 1-ethyl-1-nitrosourea (ENU). The aim of these experiments was to compare mutation susceptibility between the endogenous p53 gene and the integrated LacZ gene in the transgenic mouse. ENU-treated and control testes were analysed 102 days after treatment; a total of 297 RSM analyses were performed on ENU-treated and untreated testis DNA. Ten mutational events were detected in the p53 gene (exon 5 and intron 8), two of which occurred in untreated animals and probably represent spontaneous events. Only a single mutation was detected in the LacZ gene of an ENU-treated animal by the RSM assay. Thus the RSM assay can readily detect ENU-induced mutations in the p53 gene, but not in the LacZ transgene. Comparison of the LacZ RSM mutation data with results from a previous study of identically dosed MutaMice in the transgenic selection assay [Ashby,J., Gorelick,N.J. and Shelby,M.D. (1997) Mutat. Res., 388, 111–122] showed that LacZ mutations were far more readily recovered with the MutaMouse transgenic selection assay than by RSM analysis. The reason for the relative inability of the RSM assay to detect LacZ mutations may be the smaller target size of the RSM analysis compared with the transgenic selection assay (16 bases compared with 3000 bases). Taking into account the different target sizes by calculating the mutation frequency per base allowed the RSM data regarding p53 and LacZ to be compared with previously published data from transgenic selection assays. These studies demonstrated that the p53 mutations were present at mutation frequencies (per base) 5- to 70-fold higher than the LacZ gene mutations. In addition, the LacZ mutation frequency per base found in the RSM was an order of magnitude higher than that found in the transgenic selection assay. The transgenic selection assay is more sensitive per locus (due to the larger target of the LacZ gene), as evidenced by ability to detect ENU-induced testes mutations readily.
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Introduction |
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The occurrence of mutations is an important step in the development of cancer and heritable genetic diseases (Venkatachalam and Donehower, 1998
). Assays for measuring mutations in vivo in mammalian tissues are important in mutation research and for the assessment of mutagenic hazards to humans. Transgenic selection assays provide a unique opportunity to study the mechanisms of mutagenicity in different somatic and germinal tissues in vivo (Gossen et al., 1989; Kohler et al., 1990; Hoorn et al., 1993). The transgenic selection assay is based on the use of chromosomally integrated shuttle vectors containing reporter genes. These reporter genes can readily be recovered from genomic DNA by in vitro packaging and inspected in Escherichia coli to identify gene mutations which originated in the mouse (Vijg and van Steeg, 1998).
MutaMouse (Gossen et al., 1989) and BigBlue (Kohler et al., 1990) are the two best-known examples of the several transgenic selection assays currently available (Gossen and Vijg, 1993). For the mutational target, both systems use chromosomally integrated 
shuttle vector containing a bacterial transgene. The MutaMouse uses the E.coli LacZ gene while the BigBlue uses the E. coli LacI gene as a mutational target.
The MutaMouse transgenic selection assay was constructed by micro-injection of the bacteriophage gt10–LacZ shuttle vector into fertilized CD2 mouse oocytes. The size of the whole construct is ~47 000 bp and the LacZ gene contains 3126 bp (Gossen et al., 1989). The MutaMouse transgenic strain contains the gt10–LacZ construct in each cell, integrated at a single site as a head-to-tail concatemer in the B region of mouse chromosome 3 (Blakey et al., 1995). About 40 copies of the LacZ gene are available per haploid genome as potential targets for mutation analysis. The entire gt10–LacZ inserts have remained stably integrated in the mouse genome for many generations (Myhr, 1991). Transgenic selection assays are performed by isolating DNA from target tissues, packaging the transgene in vitro and infecting E.coli to detect mutants by phenotypic selection. Since the sequence of the LacZ gene is known, mutagenic events detected at this locus can be defined precisely at the molecular level.
It is assumed that mutations at transgenic loci can accurately reflect mutations at endogenous loci. However, differences between the endogenous and transgenic targets could result in different mutation frequencies. Firstly, the inserted transgenes are heavily methylated and non-expressed, which may result in different repair activities compared with a transcribed locus (Bohr et al., 1987). Secondly, the transgenes are usually present in multiple tandem copies. This may increase the likelihood of recombination repair (Skopek et al., 1995; Cosentino and Heddle, 1999). Hence, to assess the validity of transgenic selection assays, it is valuable to compare mutations in endogenous genes and transgenes. The results of previous comparative studies have been inconsistent and depend on many factors, such as the nature of mutagen, treatment protocol, route of mutagen administration and sampling time (Tao et al., 1993; Shaver-Walker et al., 1995; Manjanatha et al., 1998; Cosentino and Heddle, 1999). For example, Cosentino and Heddle (1999) found that the seven agents [i.e. ENU, B(a)P, BrdU, EMS, MMS, MNU and MMC] induced similar observed mutation frequencies at the endogenous Dlb-1 locus and the LacZ locus in MutaMouse after acute exposure. However, the assay's sensitivity (i.e. its ability to resolve an induced response from background) was lower for the LacZ locus than for the endogenous Dlb-1 locus. Similarly, ENU-induced mutation frequences were similar between the endogenous Hprt locus and the LacI transgene in splenocytes after acute exposure (Skopek et al., 1995), but the assay was less sensitive at the LacI locus. In contrast, X-rays induced different responses at the Dlb-1 locus and the LacZ locus (Tao et al., 1993) and the Dlb-1 locus and the LacI transgene have been shown to respond differently to chronic ENU treatment (Shaver-Walker et al., 1995).
Here we compare the mutagen sensitivity of the LacZ gene and the endogenous p53 gene in the MutaMouse using a genotypic mutation test, namely the restriction site mutation (RSM) assay (Steingrimsdottir et al., 1996; Jenkins et al., 1999). In addition, the mutagenicity of ENU in the LacZ locus, as detected by RSM analysis, was compared with previously published data from a transgenic selection assay on ENU's mutagenicity in identically dosed animals. In our study, the p53 gene was selected as a target gene because it plays a critical role as a guardian of the genome and is important for cell cycle checkpoint and apoptosis pathways (Yonish Rouach et al., 1991; Lane, 1992). Also, the p53 gene has been found to be mutated with high mutation frequencies in a wide variety of human cancers (Hollstein et al., 1996).
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Animals and husbandry
The transgenic mouse strain used in this study was MutaTMMouse (40.6). Nine male MutaTMMouse animals were used. The mice were ear punched with a unique number. Animals were housed in solid bottom cages for the duration of the experiment. Animals received RM1 (Special Diet Services Ltd.) and water ad libitum.
Animal dosing
The transgenic MutaTMMouse strain (40.6) was dosed at Zeneca Central Toxicology Lab (Alderley Park, Macclesfield, Cheshire, UK). The study consisted of five control animals and four treated animals. 1-Ethyl-1-nitrosourea (ENU) (Sigma) was dissolved in phosphate buffered saline (PBS) and administered in a single intraperitoneal injection at a dose of 150 mg/kg. The control group received i.p. injection of saline (10 ml/kg). Subsequently, animals were killed at 102 days post treatment. The germ cells in the testes 102 days post treatment were developed from stem cells exposed to ENU (Katoh et al., 1994). The testes were removed and stored at –70°C until DNA extraction was performed.
DNA extraction
DNA was extracted from the MutaTMMouse testes using the high salt method of Miller et al., (1988). DNA concentration and purity were analysed by spectrophotometry at 260/280 nm. The concentration was adjusted to 0.1 mg/ml and the DNA was stored at –20°C.
Restriction site mutation analysis
RSM analysis was performed on 1 µg aliquots of ENU-treated and untreated DNA using five endogenous p53 regions, namely exon 4, exon 5, exon 7, intron 6 and intron 8, as well as the transgenic LacZ gene region as described previously (Jenkins et al., 1999). After overnight digestion of the DNA with the test restriction enzyme, the uncut molecules were amplified by PCR and then cut again with the same restriction enzyme. Enzyme-resistant RSM products were identified by polyacrylamide gel electrophoresis. The PCR amplifications were performed with 2.5 U of Taq DNA polymerase (Promega) in `thermo' buffer (10 mM Tris–HCl pH 8.8, 50 mM KCl, 0.1% Triton X-100) with 1.5 mM MgCl2, 100 mM each dNTP and 10 pmol of each PCR primer. The final reaction volumes were adjusted to 50 µl with distilled deionized water. The conditions used for the amplification of DNA in exon 4, exon 5 and intron 6 have been detailed previously (Jenkins et al., 1997, 1998, 1999). The thermal profile for exon 7 of p53 was as follows: (i) denaturing at 94°C for 30 s; (ii) annealing at 59°C for 20 s; and (iii) extension at 72°C for 30 s; 33 thermal cycles were performed. The thermal profile for p53 intron 8 and LacZ gene regions were similar, except that the annealing temperature was 58°C and 31 cycles were performed for intron 8. For the LacZ gene, the annealing temperature was 63°C and 29 cycles were performed. Table I summarizes the PCR primers used and restriction sites analysed by the RSM assay. In the case of the p53 gene, nine restriction enzymes were used altogether, resulting in 44 bases being screened for mutations. For LacZ, four restriction enzymes were used and the number of bases screened was 16.
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Sequencing resistant RSM products to determine mutation induction
ENU-induced mutations were characterized by sequencing the restriction enzyme-resistant PCR products from the endogenous p53 target regions and transgenic LacZ gene using an ALF DNA sequencer (Pharmacia, Uppsala, Sweden).
Estimation of the mutation frequency of resistant RSM products
MutaMouse genomic DNA was diluted to give a series of 50, 102, 103 and 104 copies of the genome per 10 µl. This dilution series (10 µl samples) was then amplified alongside the RSM samples (using the same PCR master mix and the same thermal cycler). Theoretically, the efficiency of amplification should have been the same in all the tubes (Ballagi-Pordany et al., 1991; Nicoletti and Sassy-Prigent, 1996). Heterologous DNA, which ought to be added to the mouse DNA to maintain identical DNA concentrations, was not added in this study, so this is a source of potential variation. Enzyme-resistant PCR products were quantified by Sybr Gold nucleic acid gel staining of polyacrylamide gels (Molecular Probes) using a Gel Doc 2000 image analyser (Bio-Rad). Figure 1 shows a representative RSM gel showing two mutated bands alongside the external standard. The initial copy number of the mutation in genomic DNA was deduced by fitting the band intensity of the resistant RSM product to a standard curve obtained from the graph of the band intensity of the amplified DNA dilution series against copy number. The number of copies of mutant DNA calculated to be present before amplification was divided by the number of copies of the target gene analysed in each RSM reaction (3 x 105 in the case of the p53 gene) to give the mutation frequency. In calculating the LacZ mutation frequency, the calculated mutation frequency was further divided by 40 because there are 40 copies of LacZ in a single germ cell. Background mutation frequencies were estimated to be <3.3 x 10–5 (10/3 x 105) where 10 represents the minimum number of copies capable of producing a band on the RSM gel. The mutation frequencies were adjusted after sequencing to take into account amplified wild-type DNA in the enzyme-resistant products. Mutation frequencies are displayed for replicate samples, as a range of values. Mutation frequencies per base pair were calculated by dividing the RSM mutation frequencies by the number of bases in the restriction site used. For mutation frequencies obtained by the transgenic selection assay, the values were divided by 2000 (because there are 3000 bases in the gene, but only changes in the first two bases of a codon alter the amino acid and would be detectable by a phenotypic assay).
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Mutations detected in the endogenous p53 gene by RSM analysis
The results obtained from the RSM analyses of ENU-treated mouse testes are shown in Table II
. A total of 207 RSM analyses were performed on the p53 gene of MutaMouse, 115 on untreated testes and 92 on ENU-treated testes. Eight mutations were detected in ENU-treated testes samples and two mutations were also detected in untreated testes samples. The detection of eight mutations from 92 analyses on ENU-treated samples compared with two from 115 analyses of untreated samples is a statistically significant increase according to the chi-squared test (P < 0.05). These results demonstrate that the RSM assay can detect ENU-induced mutations in the endogenous p53 gene of germ cells 102 days after treatment. The ENU-induced mutations were only detected in exon 5 (four mutations) and intron 8 (four mutations) of the p53 gene. The detected mutations comprised three GC
AT transitions, two GCTA transversions, two ATGC transitions and one ATTA transversion. Given that guanine and thymine are the known targets of ENU, it is possible to determine upon which strand the mutations arose; in this case, five mutations arose on the transcribed strand and three on the non-transcribed strand. Hence there was no evidence of transcription-coupled repair in these samples, contrasting with previous data on testis mutations detected in ENU-treated CD-1 mice (Jenkins et al., 1998). Another difference between the findings of these two studies is that the mutation hotspot in the p53 gene of ENU-treated CD-1 mouse testes was shown in intron 6 (Jenkins et al., 1998), whereas no mutations were detected in intron 6 in this study.
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The highest mutation frequencies of ENU-treated samples ranged from 1.3 x 10–3 (HaeIII, exon 5) to 1 x 10–3 (RsaI, intron 8) to 1 x 10–4 (NcoI, exon 5). The background mutation frequencies were estimated to be <3.3 x 10–5 per analysis. Spontaneous mutations were only detected in the intron 8 region of the endogenous p53 gene. This is consistent with previous studies showing that intron regions are generally more mutable than the exon regions (Jenkins et al., 1997
, 1998). The spontaneous mutations comprised one GCAT transition (at a CpG site) and one ATGA transition, at a mutation frequency of 5.2 x 10–4. Table III shows the range of p53 mutation frequencies as mutation frequency per base (the mutation frequency divided by the number of bases in the restriction site analysed).
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Mutations detected in the transgenic LacZ gene by RSM analysis
Ninety RSM analyses were performed in the LacZ gene; 40 of these were on ENU-treated testis samples and 50 on untreated samples. Only a single mutation was detected in an ENU-treated sample by RSM analysis; no mutations were detected in untreated samples. The only mutation detected in the LacZ gene was a GC
AT transition, which was the same type as the most prevalent mutation in the endogenous p53 gene. The mutation frequency of this LacZ mutation was estimated to be 1.8 x 10–5; the estimated background mutation frequency was <8.3 x 10–7. Table III
shows the mutation frequencies per base of the LacZ mutations detected here and those obtained by previous transgenic selection assays. From Table III, it is evident that the LacZ mutation frequencies per base, obtained by the RSM assay here, were an order of magnitude higher than the corresponding results from the transgenic selection assay. However, the p53 mutation frequencies per base were substantially higher than those of LacZ. |
Discussion |
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These results demonstrate that the RSM assay detected ENU-induced mutations in MutaMouse testes 102 days after exposure. ENU-induced mutations were detected in the endogenous p53 gene region and, to a lesser extent, in the transgenic LacZ gene by RSM analysis. Spontaneous mutations were only detected in the intron 8 region of the endogenous p53 gene, supporting previous data on the increased mutability of intron regions (Jenkins et al., 1997
, 1998). This study confirmed that ENU was mutagenic to mouse spermatogonia cells, but it should be noted that a high dose (150 mg/kg) of ENU was employed here to achieve that mutagenicity.
The predominant mutations detected after ENU treatment were GCAT transitions (44%), followed by ATGC transitions (22%), GCTA transversions (22%) and an ATTA transversion (11%). This implied that O6-ethylguanine was the main cause of ENU-induced testis mutation. This result is consistent with the results of previous studies from this laboratory (Jenkins et al., 1998). It is interesting to note that the ENU-induced p53 mutations detected here by the RSM assay differed from mutation spectra for the LacZ gene reported in previous studies using transgenic selection assays. Douglas et al. (1995) reported that ATTA transversions were the most prevalent in ENU-treated testes of the MutaMouse by use of the transgenic selection assay (42%), followed by GCAT transitions (33%) and ATGC transitions (16%). This difference might be partly due to the fact that the restriction sites selected for our RSM study consisted of 77% GC bases. If normalized to 50% GC content, 62% of detected mutations were at AT bases (40% ATGC, 22% ATTA, 25% GCAT and 15% GCTA), perhaps suggesting that O2- and O4-ethylthymine were formed in this study. There still remain differences between this RSM study and the previous transgenic selection study of Douglas et al. (1995) with regard to induced mutation spectra. It should be noted. however, that the data presented here are based on only a small number of mutational events.
The LacZ transgene contained far fewer RSM-detectable mutations than the endogenous p53 gene (one mutation in 40 assays compared with eight in 92, respectively). The p53 mutation frequencies per base were 5- to 70-fold higher than the values obtained in LacZ by RSM. By calculating the LacZ mutation frequency per base, the values obtained in this study using RSM analysis were found to be an order of magnitude higher than previously published results of studies using the transgenic selection assay, when displayed as mutation frequency per base (Table III). Therefore, the main finding of this study is that p53 gene mutations were more readily detectable than LacZ mutations when employing the RSM assay and the mutation frequency data (per base) supports this. Whether this reflects the greater mutability of the p53 gene compared with the LacZ transgene can only be answered by further study. Hence, the RSM assay appears to be more sensitive than the transgenic selection assay in this instance, perhaps because of its ability to detect silent mutations as well as phenotype-altering mutations. However, it should be noted that given the much larger target that the LacZ gene offers in the transgenic selection assay, it will be more sensitive per locus, as is evidenced by the recovery of many ENU-induced testis mutations in the transgenic selection assay, but not in the RSM assay here. It should also be pointed out that the assumption that there are 2000 bases in the LacZ gene capable of being recovered by the transgenic selection assay when mutated may be an overestimation which may alter the comparison of the mutation frequency data. However, in this instance it does provide a useful method of comparing the sensitivity of each assay.
We have investigated the mutagenicity of ENU to Muta Mouse germ cells, employing the RSM assay, and have compared the results to those available for transgenic selection studies where mice were identically dosed. It is difficult to speculate on the relative susceptibility of endogenous genes and transgenes to ENU, due to the paucity of RSM data on recovered LacZ mutations. ENU undoubtedly induces mutations in the LacZ transgene as is evidenced by the transgenic selection data, but due to the nature of the transgene, it is not easy to identify these mutations, except by use of the transgenic selection assay (which, because of its large target size, remains more sensitive per locus than the RSM assay). The lack of RSM-detectable mutations in the LacZ gene compared with the endogenous p53 gene may be relevant in comparing locus susceptibility to mutation.
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Acknowledgments |
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The work described here was supported in part by grants from the UK Biological and Biotechnologies Research Council and the Environmental Program of the European Union. We are grateful to AstraZeneca CTL laboratories for supplying the ENU-dosed MutaMice.
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Notes |
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4 To whom correspondence should be addressed. Email: g.j.jenkins{at}swansea.ac.uk
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Ashby,J., Gorelick,N.J. and Shelby,M.D. (1997) Mutation assays in male germ cells from transgenic mice: overview of study and conclusions. Mutat. Res., 388, 111–122.[ISI][Medline]
Ballagi-Pordany,A., Ballagi-Pordany,A. and Funa,A. (1991) Quantitative determination of mRNA phenotypes by the polymerase chain reaction. Anal. Biochem., 196, 89–94.[ISI][Medline]
Blakey,D.H., Douglas,G.R., Huang,K.C. and Winter,H.J. (1995) Cytogenetic mapping of gt10 LacZ sequences in the transgenic mouse strain 40. 6 (MutaTMMouse). Mutagenesis, 10, 145–148.
Bohr,V.A., Phillips,D.H. and Hanawalt,P.C. (1987) Heterogeneous DNA damage and repair in the mammalian genome. Cancer Res., 47, 6426–6436.[ISI][Medline]
Cosentino,L. and Heddle,J.A. (1999) A comparison of effects of diverse mutagens at lacZ transgene and Dlb-1 locus in vivo. Mutagenesis, 14, 113–119.
Douglas,G.R., Jiao,J., Gingerich,J.D., Gossen,J.A. and Soper,L.M. (1995) Temporal and molecular characteristics of mutations induced by ethylnitrosourea in germ cells isolated from seminiferous tubules and in spermatozoa of LacZ transgenic mice. Proc. Natl Acad. Sci. USA, 92, 7485–7489.
Douglas,G.R., Gingerich,J.D., Soper,L.M. and Jiao,J. (1997) Toward an understanding of the use of transgenic mice for the detection of gene mutations in germ cells. Mutat. Res., 388, 197–212.[ISI][Medline]
Gossen,J.A. and Vijg,J.A. (1993) Transgenic mice as model systems for studying gene mutations in vivo. Trends Genet., 9, 27–31.[ISI][Medline]
Gossen,J.A., Deleeuw,W.J.F., Tan,C.H.T., Zwarthoff,E.C., Berends,F., Lohman,P.H.M., Knook,D.L. and Vijg,J. (1989) Efficient rescue of integrated shuttle vectors from transgenic mice: a model for studying mutations in vivo. Proc. Natl Acad. Sci. USA, 86, 7971–7975.
Hollstein,M., Shamer,B., Greenblatt,M., Soussi,T., Hovig,E., Montesano,R. and Harris,C.C. (1996) Somatic point mutations in the p53 gene of human tumours and cell lines: update compilation. Nucleic Acids Res., 24, 141–146.
Hoorn,A.J.W., Custer,L.L., Myhr,B.C., Brusick,D., Gossen,J. and Vijg,J. (1993) Detection of chemical mutagens using MutaTMMouse: a transgenic mouse model. Mutagenesis, 8, 7–10.
Jenkins,G.J.S., Chaleshtori,M.H., Song,H. and Parry,J.M. (1998) Mutation analysis using the restriction site mutation (RSM) assay. Mutat. Res., 405, 209–220.[ISI][Medline]
Jenkins,G.J.S., Mitchell,I.de G. and Parry,J.M. (1997) Enhanced restriction site mutation (RSM) analysis of 1,2-dimethylhydrazine induced mutations, using endogenous p53 intron sequences. Mutagenesis, 12, 117–124.
Jenkins,G.J.S., Suzen,H.S., Sueiro,R.A. and Parry,J.M. (1999) The restriction site mutation assay: a review of the methodology development and the current status of the technique. Mutagenesis, 14, 439–448.
Katoh,M., Inomata,T., Horiya,N., Suzuki,F., Shida,T., Ishioka,K. and Shibuya,T. (1994) Studies on mutations in male germ cells of transgenic mice following exposure to isopropyl methanesulfonate, ethylnitrosourea or X-ray. Mutat. Res., 341, 17–28.[ISI][Medline]
Kohler,S.W., Provost,G.S., Kretz,P.L., Dycaico,M.J., Sorge,J.A. and Short,J.M. (1990) Development of a short-term, in vivo mutagenesis assay: the effects of methylation on the recovery of lambda phage shuttle vector from transgenic mice. Nucleic Acids Res., 18, 3007–3013.
Lane,D.P. (1992) Cancer: p53, guardian of the genome. Nature, 358, 15–16.[Medline]
Manjanatha,M.G., Shelton,S.D., Aidoo,A., Lyn-Cook,L.E. and Casciano,D.A. (1998) Comparison of the in vivo mutagenesis in the endogenous Hprt gene and the LacI transgene of BigBlue rats treated with 7,12-dimthylbenz[a]anthracene. Mutat. Res., 401, 165–178.[ISI][Medline]
Mattison,J.D., Penrose,L.B. and Burlinson,B. (1997). Preliminary results of ethylnitrosourea, isopropyl methanesulphonate and methyl methane- sulphonate activity in the testis and epididymal spermatozoa of MutaTMMice. Mutat. Res., 388, 123–127.[ISI][Medline]
Miller,S.A., Dykes,D.D. and Polesky,H.F. (1988) A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res., 16, 1215.
Myhr,B.C. (1991) Validation studies with MutaTMMouse: a transgenic mouse model for detecting mutations in vivo. Environ. Mol. Mutagen., 18, 308–315.[ISI][Medline]
Nicoletti,A. and Sassy-Prigent,C. (1996) An alternative quantitative polymerase chain reaction method. Anal. Biochem., 236, 229–241.[ISI][Medline]
Renault,D., Brault,D. and Thybaud,V. (1997) Effect of ethylnitrosourea and methyl methanesulfonate on mutation frequency in MutaTMMouse germ cells (seminiferous tubles cells and epididymis spermatozoa). Mutat. Res., 388, 145–153.[ISI][Medline]
Shaver-Walker,P.M., Urlando,C., Tao,K.S., Zhang,X.B. and Heddle,J.A. (1995) Enhanced somatic mutation rates induced in stem cells by low chronic exposures to ethylnitrosourea. Proc. Natl Acad. Sci. USA, 92, 11470–11474.
Skopek,T.R., Kort,K.L. and Marino,D.R. (1995) Relative sensitivity of endogenous hprt gene and lacI transgene in ENU-treated Big BlueTM B6C3F1 mice. Environ. Mol. Mutagen., 26, 9–15.[ISI][Medline]
Steingrimsdottir,H., Beare,D., Cole,J., Leal,J.F.M., Kostic,T., Lopez-Barea,J., Dorado,G. and Lehmann,A.R. (1996) Development of new molecular procedures for the detection of genetic alteration in man. Mutat. Res., 353, 109–121.[ISI][Medline]
Tao,K.S., Urlando,C. and Heddle,J.A. (1993) Comparison of somatic mutation in a transgenic versus host locus. Proc. Natl Acad. Sci. USA, 90, 10681–10685.
Tinwell,H., Lefevre,P., Williams,C.V. and Ashby,J. (1997) The activity of ENU, iPMS and MMS in male mouse germ cells using the MutaTMmouse positive selection transgenic mutation. Mutat. Res., 388, 179–185.[ISI][Medline]
Venkatachalam,S. and Donehower,L.A. (1998) Murine tumour suppressor models. Mutat. Res., 400, 391–407.[ISI][Medline]
Vijg,J. and van Steeg,H. (1998) Transgenic assays for mutations and cancer: current status and future perspectives. Mutat. Res., 400, 337–354.[ISI][Medline]
Yonisch Rouach,E., Resnitsky,D., Lotem,J., Sachs,A., Kimuchi,A. and Oren,M. (1991) Wild type p53 induced apoptosis of myeloid leukaemic cells that is inhibited by interleukin 6. Nature, 352, 345–347.[Medline]
Received on May 29, 2000; accepted on September 11, 2000.
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