Transgenic Mice and Age-Related Mutations JAN VIJG,sb WILJO J. F. DE LEEUW: GEORGE R. DOUGLAS: AND JAN A. GOSSEN'T~ aMedscand Ingeny P.O. Box 685 2300 AR Leiden, the Netherlands bHarvard Medical School Beth Israel Hospital GerontologyDivision Boston, Massachusetts 02215 CEnvironmentalHealth Centre Tunney's Pasture Ottawa, Ontario KIA OL2, Canada The concept of random genetic changes as a fundamental cause of senescence is still subject to controversy despite the fact that some somatic mutation theories of aging were formulated decades ago, including the potential relationship to cancer and other age-related diseases such as atherosclerosis and Alzheimer's disease.'-' Although there is ample experimental evidence for an increase in large structural genomic changes (such as chromosomal aberrations) with age in both man5*6and a ~ ~ i m agene l , ~ . mutations ~ have thus far been inaccessible for in vivo studies, with the exception of selectable genes such as hypoxanthine guanine phosphoribosyl transferase (HPRT). Upon inactivation, this X-linked gene confers resistance to purine analogs such as 6-thioguanine?J0 From initial experiments on human lymphocytes involving the WRTgene as the selectable target, the mutation frequencies found by different investigators varied from between and I t to 10-4.12,13Furthermore, although Strauss and Albertini I2 did not observe any increase in the average mutant frequency with age, Evans and Vijayala~mi,'~ Morley et al.," and Trainor et all4 reported an age-related increase of a few percentages per year on average. These discrepancies are likely due to inadequate stringency of ~election.'~ In later studies the mutant nature of the 6-thioguanine-resistant cells was confirmed by expansion of the clones, which allowed the heritable nature of the mutations and their characterization to be proved. It is now clear that mutant frequencies range from about 0.6 x in newborns to about 16 x in aged twins, while young adults have a mutant frequency of about 6 x 10-6.15Age-related increases in mutant frequencies in human lymphocytes were also reported by Morley and co-workers, using the HLA-A system.16 Thus far, only one study has reported on the frequency of nonhuman primary cells mutated at the HPRT locus. In this study, primary cells from mouse kidney and skeletal muscle were clonally assessed for 6-thioguanine resistance.17 Although the resistant clones were not characterized with respect to the molecular nature of the mutations, HPRT deficiency was convincingly dem~nstrated.'~ The mutant frequencies observed were in the order of 10-5-10-4. The investigators considered their results on 15 mice of different ages as evidence for the absence of an age-related 26
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increase in mutant frequency. However, in view of the rather high interindividual variation, these findings need to be confirmed. In most studies a considerable part of the mutants consists of rearrangements. For example, Nalbantoglu et al., analyzing the APRT gene in hamster cells, found that about 10% of the mutations inactivating this gene were deletions or insertions of more than 30 base pairs occurring between short direct sequence repeats. Analysis of the much larger HPRT gene in human T-cells in vivo showed that among 30 HPRT-deficient mutants obtained from 7 normal control individuals, 6 were deletions of up to 38 kilobases.Iy In human adults, structural gene alterations were observed in 10-20%z0 and 57?hZ1of the HPRT mutants studied. Interestingly, in newborns the spectrum appeared to be quite different. McGinniss et al.z2found gross structural alterations in 81% of the mutants. Using T-cell receptor rearrangements as a marker, these authors showed that a considerable part of these mutations had independently arisen in prethymic stem cells, which is in contrast to the mostly postthymic T-lymphocyte mutants in adults.2z In summary, from the limited studies thus far performed on gene mutations in somatic cells in relation to donor age, one can conclude that there is an age-related increase in mutant frequency in human lymphocytes. This increase seems most dramatic between newborns and young adults. Before any definite conclusions can be drawn, more data, especially on individuals of extremely advanced age, are necessary. In addition, in view of differences in the nature of the mutants between newborns and adults, mutation spectra for groups of individuals at different age levels are highly desirable. The HPRT and comparable tests can only be used to obtain data sets on mutation spectra of the target gene in cells that can easily be isolated and clonally assessed in vitro. Other disadvantages of these systems for analyzing spontaneous and induced mutations in relation to in vivo age are that they are time consuming, only a limited number of cell types can be analyzed, and mutations observed may not always reflect mutation frequencies in the tissue of origin.23 To directly analyze different organs and tissues for the presence of low-frequency DNA mutations, other systems have been developed. A procedure based on the use of denaturing gradient gel electrophoresis to separate mutant and nonmutant PCR-amplified target sequences was proposed by Vijg and Uitterlinden.z4 Recently, Thilly and coworkersz5 demonstrated that it is possible to separate in denaturing gradient gels heteroduplexed mutant HPRT sequences obtained from uncloned, complex populations of human B-lymphocytes treated with large doses of different mutagens and cultured in the presence of 6 - t h i o g ~ a n i n e Application .~~ of this method for studying mutations occurring at low frequency, however, has not been established yet. Despite the use of high-fidelity polymerases such as T4 and T7 and alterations in the experimental conditions,26the PCR amplification step was found to introduce too many misincorporations (J. A. Gossen, unpublished data), thereby overshadowing spontaneous mutation frequencies. Jeffreys et aL2' developed a method to specifically amplify and characterize in vivo deletion mutations at the D1S8 minisatellite locus in human genomic DNA isolated from blood and sperm. This is the first method that monitors deletion-type mutations directly in tissue DNA. However, point mutations and mutations in other not so unstable loci go undetected in this method, which demonstrates the need to develop additional methods. As yet, such methods are not available, and mutation analysis in various organs and tissues in humans is therefore not possible. Transgenic animal technology was recently applied in developing improved mutagenesis testing systems. The approaches used include the use of shuttle vectors harboring a bacterial marker gene acting as a target for mutagenesis.z8~zy The
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applicability of such a system to detect induced mutations in vivo was demonstrated by Gossen et al.,% Hoorn et al.,30and Kohler et al.29Studies with transgenic mice on the mutagenicity of ethylnitrosourea have demonstrated that these systems are capable of detecting organ-specific and dose-dependent mutation frequencies in liver, brain, bone marrow, spleen, and testis, even when small doses were administered.% In addition, this approach allows easy characterization of mutation spectra in different organs and tissues. DNA sequence analysis of mutant lacZ genes isolated from brain DNA of mice treated with ethylnitrosourea indicated predominantly base pair substitutions.% Compared to in vitro assays, in vivo assays, like the transgenic mouse systems, more accurately account for the physiological and metabolic parameters that influence the mutagenic potential of a compound. Assuming the integration site of the vector to be more or less random, the transgenic mouse models also allow for comparative analysis of different genomic sites for their susceptibility to mutation induction. The relevance of such mutational hotspots, the presence of which could already be inferred from studies with cultured cells and from genetic studies, for the aging process is obvious; depending on the functional status of the D N A sequences involved, genetic damage could accumulate much faster than hitherto foreseen on the basis of the adopted (low) rate of spontaneous mutation induction. Here we report a mutational hotspot on the mouse X chromosome.
MATERIALS AND METHODS
The first series of transgenic mouse mutation models we recently constructed harbors the bacterial lacZ gene as a target for mutagenesis in all organs and tissues including the germ cells. The lacZ gene was cloned into the bacteriophage lambda gt-10 vector, allowing efficient rescue from total genomic mouse DNA by means of in vitro packaging. Mutation frequencies are determined from total genomic DNA, isolated as previously described, as the ratio between colorless (mutated) and blue (nonmutated) plaques% (FIG. 1). A number of strains have been constructed, all but one of which could be characterized by a low background mutation frequency. The exception is strain 35.5,which was used in the present study.
RESULTS AND DISCUSSION
To compare spontaneous mutation frequencies at three different loci, lambdagtlOLacZ shuttle vectors were recovered from liver and brain DNA of mice from three transgenic mouse strains: 20.2 (80 copies per haploid genome), 35.5 (15 copies per haploid genome), and 40.6 (40 copies per haploid genome). The results obtained with transgenic mouse strains 20.2 and 40.6 indicate spontaneous mutation frequencies in liver and brain DNA of between 1 x loT6and 1x (TABLE1). Such mutation frequencies are in the same range as the spontaneous mutation frequencies found in animal and human T-lymphocytes using the HPRT clonal assay.17~zo*z1 Interestingly, considerably higher spontaneous mutation frequencies were observed in mice from strain 35.5,with mean values of about for liver D N A and 9.2 x 7.9 x for brain DNA. A significantly higher spontaneous mutation frequency was observed in brain DNA of homozygote animals compared to hemizygotes (TABLE 1). For brain DNA isolated from hemi- or
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fertilized egg with pronuclei injection pipette holding p i p
3
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W
I
oviductal implantation
4
7
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viral vector DNA carrying lac2 as a target gene for mutations
9
empty virus capsule
DNA isolation
J
plating on IacZ- bacteria with X-Gal and IPTG
rescue o f integrated
n o mutant 0 w i l d type
FIGURE 1. Schematic depiction of the use of bacteriophage lambda shuttle vectors integrated in the genome of (transgenic) mice for mutation analysis in vivo.
homozygote mice of strain 35.5, the spontaneous mutation frequencies were significantly different (p < 0.05),as determined by Student’s t test.31 To investigate the nature of the increased spontaneous mutation frequency in the lambda-gtl0LacZ concatemer in this specific transgenic mouse strain, knowledge concerning its chromosomal position is essential. Indeed, the primary and/or higher order structure of the lambda-gtl0LacZ concatemer and/or its flanking sequences may well be the underlying cause of the increased susceptibility to mutagenesis. The
TABLE1. Spontaneous Mutation Frequencies in Lambda-gt lOLacZ Rescued from Liver and Brain DNA of Three Different Transgenic Mouse Strains Strain 20.2 40.6 35.5“ 35.5*
Orean Liver (3) Brain (3) Liver (4) Brain (4) Liver (17) Brain (19) Liver (5) Brain (9)
“Hemizygote. bHomozygote.
No. of Plaques/ No. of Animals Analvzed 6561883 5211356 258/562 1.02210 7581936 598/938 288/136 4051568
No. of Mutants Analvzed 1 0 1 1 62 29 21 63
Mutation Freauencv ( X 0.1 nd 0.7 0.1 8.1 2 6.1 5.1 2 2.4 1.3 2 3.5 15.5 2 8.8
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FIGURE 2. Southern blot analysis of tail DNA of mice obtained from crossings between hemizygous 35.5 male and female (A) mice and from crossings between 35.5 male and female mice not possessing the 35.5 lambda-gtl0LacZ vector (B).Deletions/duplications are indicated by arrows (a, b, c, and d).
finding that after crossings between hemizygote mice the male progeny were never homozygote, and that after crossings between hemizygote male mice and nontransgenic female mice the lambda-gtl0LacZ concatemer was transmitted to the daughters only indicated that the lambda-gtl0LacZ concatemer in strain 35.5 is located on the X chromosome (Gossen et ~ 1 . ~ ~ ) . Southern blot analysis of tail DNA of mice, obtained from crossings between hemizygote male (XzY) and female (XzX) 35.5 mice and from crossings between hemizygote 35.5 male (XzY) and female mice not containing the 35.5 lambdagtlOLacZ concatemer (XX), revealed rearrangements of 3’ flanking regions of the lambda-gtl0LacZ concatemer (FIG.2). In addition to a strong hybridizing 1.2-kb head-to-tail fragment containing the lambda cos-site, 12 other fragments are detected by this probe (FIG.2). These fragments are due to extensive rearrangements near the 3’ site of integration, the occurrence of which during integration is typical for transgenesis.32It should be noted that after these integration-associated rearrangements have taken place, the lambda-gtl0LacZ concatemer and its 3’ and 5’ surrounding regions should be stable, as has been observed for all other transgenic mouse strains except the 35.5 strain. Among 96 mice of strain 35.5 analyzed, 9 of 57 female (15.8%) and only 1 of 39 male (3%) mice contained deletions and/or duplications in the 3’ flanking region, and two female animals contained both a deletion and a duplication. In contrast to the instability observed in the 3’ flanking region, deletions and/or duplications were not detected in the 5’ flanking region. In addition, although all other lambda-
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gtlOLacZ transgenic mouse strains were found to contain lambda parts in the regions flanking the concatemer, instabilities have never been observed. The high frequency of deletions and/or duplications observed in female mice indicates deletion and duplication events of 3' regions occurring preferentially during male meiosis. The meiotic origin of the rearrangements is indicated by our observation that the same deletions and duplications were also present in liver and brain DNA of the same mice (results not shown). To compare the kind of mutations present in lacZ genes isolated from liver and brain DNA of strains 35.5 and 40.6, 90 mutants were analyzed for the presence of large structural changes ( > 30 bp) by means of PCR analysis. Lambda primers, located near the EcoRI site of lambda-gtl0, were used to amplify the mutant facZ genes directly from colorless lambda-gtl0lacZ plaques. From TABLES 2 and 3 it can be concluded that in both strains the whole spectrum of mutational events, from deletions to point mutations, is represented. However, whereas in the 35.5 strain all
TABLE2. Sequence Analysis of Spontaneous LacZ Mutants Isolated from Liver and Brain DNA of Mice of Strain 35.5 Organ Liver
Mutant 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Brain
16 17 18 19 1 2 3 4 5 6 7 8 9
Sequence Alteration deletion G:C C:G+TA (TS) G:C+AT (TS) A T + T A (TV) G:C+TA (TV) T:A+AT (TV) T A + C G (TS) deletion 21 bp TA+C:G (TS) CG-*T:A (TS) G:C+AT (TS) deletion C:G C:G+TA (TS) G:C+AT (TS) deletion C G T A C G (TS) C:G-+TA (TS) G:C+TA (TV) C G - + T A(TS) G:C+AT (TS) deletion C:G A:T+C:G (TV) CG-+A:T (TV) C:G+AT (TV) deletion A:T C:G-+AT (TV) deletion 1046 bp C:G+A:T (TV) deletion 1046 bp C:G+TA (TS) deletion A T C:G+A:T (TS) AT-+T:A (TV) C:G+TA (TS)
Position 1524 1270 1676 1637 1721 1242 1577 1592-1613 1720 1592 1627 1626 1388 1627 1626 1562 1196 1348 1159 1708 1318 1338 1638 1520 1651 664 670-1716 664 670-1716 1399 1397 1329 993 1640
Target Sequence CGAT.TACG AACGACGG TACTAGCAG GGGnACAG GTWGGAC TGTWGTGG TCCACAAAA rn.CGCT CGTCCGGGA CTlTIJ3CTA TI'GCMTA TTTG.GAAT TGGnGCTG ITGC-WTA 'ITTG.GAAT GCTGCGCCG T A T q GAAC AATGPGCA GAAGTAGAA ACAGAGCGG GCTA.CGGC GCGTGACGC CCCWGCGA TGCCAGATG TAAC.GTCT GCTGgTAA ATAA.CAG A GCTGFTAA ATAA.CAGA GAATWTC GGGA.TGAA TGAGAGAAC AAGCIGAAG CACGTGATG
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TABLE 3. DNA Sequence Analysis of Spontaneous LacZ Mutants Isolated from the Liver of Transgenic Mice of Strain 40.6 Mutant 1 2 3 4 5 6 7 8 9
10 12 14
15 16 18 19 20 21 24 25 26 27 28
Sequence Alteration TA-C:G (TS) CG-TA (TS) TA-CG (TS) CG-TA (TS) AT-TA (TV) deletion G:C AT-CG (TV) CG-TA (TS) C:G-T.A (TS) GC+AT (TS) deletion T A deletion ATTA TA-CG (TS) CG+TA (TS) CG+TA (TS) AT-G:C (TS) G:C-AT (TS) CG-T:A (TS) G:C-AT (TS) G:C-AT (TS) TA+C:G (TS) G:C+TA (TV) AT-TA (TV) AT-+G:C(TS) TA-CG (TS) C:G-+T:A(TS) deletion A T
Position 1103 1531 1962 2743 617 1370 1096 1531 461 507 1585 1911/1912 779 2813 1529 634 2142 1196 901 820 2633 1873 1883 1602 1376 1531 2822
Target Sequence CCrCCGCAT CGCGIGCGT GCGACAACG CGA(JTGCCr GATnGGAT CACC.GATG GAGCgCAT CGCGIGCGT AAm-GGCG GCCMGACA AAAA.GGCr GllT..CCG TACCCACGG 'ITC(JTGAGC TACGIGCGC GATGGGCGG CATGATCAG TATnGAAC GAACATCGA GGTCACCAG GAAGCGGCG GACGTAAGC AAAnCCAG CTGGGGAGA AGTGCGATC CGCGTGCGT GAAA.CGGT
mutations are present in a small area of about 500 bp, the 40.6 strain has the mutations spread all over the 3,000-bp lacZ gene. Furthermore, of all transition mutations the target sequence in the 40.6 strain is clearly a methylated CpG-rich region, which is not the case with the 35.5 strain. Therefore, we conclude that the mechanism of mutation induction in the 35.5 strain differs considerably between the two genomic sites. It is well documented that the majority of basepair mutations (68%) appear to be replication dependent.33 It is therefore likely that the high spontaneous mutation frequency in strain 35.5 reflects misincorporations opposite apurinic sites by DNA polymera~es,3~ for example, as a consequence of differences in chromatin structure or the asynchronous replication of active and inactive X chromosomes during cell repli~ation.~~ The latter could be tested by crossing the 35.5 strain with Searle's translocation mice [t(X,16)] and comparing mutation frequency in females where the lambda-gtl0LacZ concatemer is on the normal or on the rearranged X chromosome. If the lambda-gtl0LacZ concatemer is indeed preferentially present on the active X chromosome, this may account for the observed difference in spontaneous mutation frequency between hemi- and homozygote brain D N A (TABLE 1) and would explain the absence of differences in mutation frequencies between hemizygote male and female mice. Our results suggest that the high spontaneous mutation frequency in liver and
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brain DNA of strain 35.5 is due to the location of the lambda-gtl0LacZ concatemer at a highly unstable chromosomal region, making it more susceptible to both germinal and somatic mutagenesis. The large differences in spontaneous mutation frequencies between the 35.5 and the other two strains may therefore indicate the presence of a mutational “hot spot” in the somatic genome that has hitherto only been found for some minisatellite A major question regarding the origin of the high number of spontaneous mutations in strain 35.5 is when these events take place. Indeed, if such high mutation frequencies would be constant, one would expect that at old age virtually all copies of the gene will have undergone a mutational change. We have currently performed only very limited studies on the effect of aging on the mutation rate. Preliminary data of mutant frequencies in liver and brain as a function of chronologic age indicate high interindividual variation, but no dramatic change with age (results not shown). In this regard it is not inconceivable that at some early stage during embryogenesis a burst of mutational activity is responsible for the high mutant frequencies observed. In any case the question as to whether this specific chromosomal site is more susceptible to an age-related increase in mutation frequency remains an intriguing one. Finally, the bacteriophage lambda-based models just described have the major disadvantages that (1) a selection against nonmutant plaques is lacking and (2) expensive packaging extracts are needed. Therefore, a new system has been developed that is based on plasmid rescue by magnetic beads and mutation detection by subsequent transformation of Gal E- Eschen’chiu coli cells.37This avoids the need for expensive in vitro packaging extracts and provides a selection step in which all nonmutants are killed. This system is most suitable for the large-scale studies on spontaneous and induced mutations that are necessary to obtain insight into the relation between somatic mutations and the aging process.
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increase in mutant frequency. However, in view of the rather high interindividual variation, these findings need to be confirmed. In most studies a considerable part of the mutants consists of rearrangements. For example, Nalbantoglu et al., analyzing the APRT gene in hamster cells, found that about 10% of the mutations inactivating this gene were deletions or insertions of more than 30 base pairs occurring between short direct sequence repeats. Analysis of the much larger HPRT gene in human T-cells in vivo showed that among 30 HPRT-deficient mutants obtained from 7 normal control individuals, 6 were deletions of up to 38 kilobases.Iy In human adults, structural gene alterations were observed in 10-20%z0 and 57?hZ1of the HPRT mutants studied. Interestingly, in newborns the spectrum appeared to be quite different. McGinniss et al.z2found gross structural alterations in 81% of the mutants. Using T-cell receptor rearrangements as a marker, these authors showed that a considerable part of these mutations had independently arisen in prethymic stem cells, which is in contrast to the mostly postthymic T-lymphocyte mutants in adults.2z In summary, from the limited studies thus far performed on gene mutations in somatic cells in relation to donor age, one can conclude that there is an age-related increase in mutant frequency in human lymphocytes. This increase seems most dramatic between newborns and young adults. Before any definite conclusions can be drawn, more data, especially on individuals of extremely advanced age, are necessary. In addition, in view of differences in the nature of the mutants between newborns and adults, mutation spectra for groups of individuals at different age levels are highly desirable. The HPRT and comparable tests can only be used to obtain data sets on mutation spectra of the target gene in cells that can easily be isolated and clonally assessed in vitro. Other disadvantages of these systems for analyzing spontaneous and induced mutations in relation to in vivo age are that they are time consuming, only a limited number of cell types can be analyzed, and mutations observed may not always reflect mutation frequencies in the tissue of origin.23 To directly analyze different organs and tissues for the presence of low-frequency DNA mutations, other systems have been developed. A procedure based on the use of denaturing gradient gel electrophoresis to separate mutant and nonmutant PCR-amplified target sequences was proposed by Vijg and Uitterlinden.z4 Recently, Thilly and coworkersz5 demonstrated that it is possible to separate in denaturing gradient gels heteroduplexed mutant HPRT sequences obtained from uncloned, complex populations of human B-lymphocytes treated with large doses of different mutagens and cultured in the presence of 6 - t h i o g ~ a n i n e Application .~~ of this method for studying mutations occurring at low frequency, however, has not been established yet. Despite the use of high-fidelity polymerases such as T4 and T7 and alterations in the experimental conditions,26the PCR amplification step was found to introduce too many misincorporations (J. A. Gossen, unpublished data), thereby overshadowing spontaneous mutation frequencies. Jeffreys et aL2' developed a method to specifically amplify and characterize in vivo deletion mutations at the D1S8 minisatellite locus in human genomic DNA isolated from blood and sperm. This is the first method that monitors deletion-type mutations directly in tissue DNA. However, point mutations and mutations in other not so unstable loci go undetected in this method, which demonstrates the need to develop additional methods. As yet, such methods are not available, and mutation analysis in various organs and tissues in humans is therefore not possible. Transgenic animal technology was recently applied in developing improved mutagenesis testing systems. The approaches used include the use of shuttle vectors harboring a bacterial marker gene acting as a target for mutagenesis.z8~zy The
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applicability of such a system to detect induced mutations in vivo was demonstrated by Gossen et al.,% Hoorn et al.,30and Kohler et al.29Studies with transgenic mice on the mutagenicity of ethylnitrosourea have demonstrated that these systems are capable of detecting organ-specific and dose-dependent mutation frequencies in liver, brain, bone marrow, spleen, and testis, even when small doses were administered.% In addition, this approach allows easy characterization of mutation spectra in different organs and tissues. DNA sequence analysis of mutant lacZ genes isolated from brain DNA of mice treated with ethylnitrosourea indicated predominantly base pair substitutions.% Compared to in vitro assays, in vivo assays, like the transgenic mouse systems, more accurately account for the physiological and metabolic parameters that influence the mutagenic potential of a compound. Assuming the integration site of the vector to be more or less random, the transgenic mouse models also allow for comparative analysis of different genomic sites for their susceptibility to mutation induction. The relevance of such mutational hotspots, the presence of which could already be inferred from studies with cultured cells and from genetic studies, for the aging process is obvious; depending on the functional status of the D N A sequences involved, genetic damage could accumulate much faster than hitherto foreseen on the basis of the adopted (low) rate of spontaneous mutation induction. Here we report a mutational hotspot on the mouse X chromosome.
MATERIALS AND METHODS
The first series of transgenic mouse mutation models we recently constructed harbors the bacterial lacZ gene as a target for mutagenesis in all organs and tissues including the germ cells. The lacZ gene was cloned into the bacteriophage lambda gt-10 vector, allowing efficient rescue from total genomic mouse DNA by means of in vitro packaging. Mutation frequencies are determined from total genomic DNA, isolated as previously described, as the ratio between colorless (mutated) and blue (nonmutated) plaques% (FIG. 1). A number of strains have been constructed, all but one of which could be characterized by a low background mutation frequency. The exception is strain 35.5,which was used in the present study.
RESULTS AND DISCUSSION
To compare spontaneous mutation frequencies at three different loci, lambdagtlOLacZ shuttle vectors were recovered from liver and brain DNA of mice from three transgenic mouse strains: 20.2 (80 copies per haploid genome), 35.5 (15 copies per haploid genome), and 40.6 (40 copies per haploid genome). The results obtained with transgenic mouse strains 20.2 and 40.6 indicate spontaneous mutation frequencies in liver and brain DNA of between 1 x loT6and 1x (TABLE1). Such mutation frequencies are in the same range as the spontaneous mutation frequencies found in animal and human T-lymphocytes using the HPRT clonal assay.17~zo*z1 Interestingly, considerably higher spontaneous mutation frequencies were observed in mice from strain 35.5,with mean values of about for liver D N A and 9.2 x 7.9 x for brain DNA. A significantly higher spontaneous mutation frequency was observed in brain DNA of homozygote animals compared to hemizygotes (TABLE 1). For brain DNA isolated from hemi- or
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fertilized egg with pronuclei injection pipette holding p i p
3
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I
oviductal implantation
4
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viral vector DNA carrying lac2 as a target gene for mutations
9
empty virus capsule
DNA isolation
J
plating on IacZ- bacteria with X-Gal and IPTG
rescue o f integrated
n o mutant 0 w i l d type
FIGURE 1. Schematic depiction of the use of bacteriophage lambda shuttle vectors integrated in the genome of (transgenic) mice for mutation analysis in vivo.
homozygote mice of strain 35.5, the spontaneous mutation frequencies were significantly different (p < 0.05),as determined by Student’s t test.31 To investigate the nature of the increased spontaneous mutation frequency in the lambda-gtl0LacZ concatemer in this specific transgenic mouse strain, knowledge concerning its chromosomal position is essential. Indeed, the primary and/or higher order structure of the lambda-gtl0LacZ concatemer and/or its flanking sequences may well be the underlying cause of the increased susceptibility to mutagenesis. The
TABLE1. Spontaneous Mutation Frequencies in Lambda-gt lOLacZ Rescued from Liver and Brain DNA of Three Different Transgenic Mouse Strains Strain 20.2 40.6 35.5“ 35.5*
Orean Liver (3) Brain (3) Liver (4) Brain (4) Liver (17) Brain (19) Liver (5) Brain (9)
“Hemizygote. bHomozygote.
No. of Plaques/ No. of Animals Analvzed 6561883 5211356 258/562 1.02210 7581936 598/938 288/136 4051568
No. of Mutants Analvzed 1 0 1 1 62 29 21 63
Mutation Freauencv ( X 0.1 nd 0.7 0.1 8.1 2 6.1 5.1 2 2.4 1.3 2 3.5 15.5 2 8.8
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FIGURE 2. Southern blot analysis of tail DNA of mice obtained from crossings between hemizygous 35.5 male and female (A) mice and from crossings between 35.5 male and female mice not possessing the 35.5 lambda-gtl0LacZ vector (B).Deletions/duplications are indicated by arrows (a, b, c, and d).
finding that after crossings between hemizygote mice the male progeny were never homozygote, and that after crossings between hemizygote male mice and nontransgenic female mice the lambda-gtl0LacZ concatemer was transmitted to the daughters only indicated that the lambda-gtl0LacZ concatemer in strain 35.5 is located on the X chromosome (Gossen et ~ 1 . ~ ~ ) . Southern blot analysis of tail DNA of mice, obtained from crossings between hemizygote male (XzY) and female (XzX) 35.5 mice and from crossings between hemizygote 35.5 male (XzY) and female mice not containing the 35.5 lambdagtlOLacZ concatemer (XX), revealed rearrangements of 3’ flanking regions of the lambda-gtl0LacZ concatemer (FIG.2). In addition to a strong hybridizing 1.2-kb head-to-tail fragment containing the lambda cos-site, 12 other fragments are detected by this probe (FIG.2). These fragments are due to extensive rearrangements near the 3’ site of integration, the occurrence of which during integration is typical for transgenesis.32It should be noted that after these integration-associated rearrangements have taken place, the lambda-gtl0LacZ concatemer and its 3’ and 5’ surrounding regions should be stable, as has been observed for all other transgenic mouse strains except the 35.5 strain. Among 96 mice of strain 35.5 analyzed, 9 of 57 female (15.8%) and only 1 of 39 male (3%) mice contained deletions and/or duplications in the 3’ flanking region, and two female animals contained both a deletion and a duplication. In contrast to the instability observed in the 3’ flanking region, deletions and/or duplications were not detected in the 5’ flanking region. In addition, although all other lambda-
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gtlOLacZ transgenic mouse strains were found to contain lambda parts in the regions flanking the concatemer, instabilities have never been observed. The high frequency of deletions and/or duplications observed in female mice indicates deletion and duplication events of 3' regions occurring preferentially during male meiosis. The meiotic origin of the rearrangements is indicated by our observation that the same deletions and duplications were also present in liver and brain DNA of the same mice (results not shown). To compare the kind of mutations present in lacZ genes isolated from liver and brain DNA of strains 35.5 and 40.6, 90 mutants were analyzed for the presence of large structural changes ( > 30 bp) by means of PCR analysis. Lambda primers, located near the EcoRI site of lambda-gtl0, were used to amplify the mutant facZ genes directly from colorless lambda-gtl0lacZ plaques. From TABLES 2 and 3 it can be concluded that in both strains the whole spectrum of mutational events, from deletions to point mutations, is represented. However, whereas in the 35.5 strain all
TABLE2. Sequence Analysis of Spontaneous LacZ Mutants Isolated from Liver and Brain DNA of Mice of Strain 35.5 Organ Liver
Mutant 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Brain
16 17 18 19 1 2 3 4 5 6 7 8 9
Sequence Alteration deletion G:C C:G+TA (TS) G:C+AT (TS) A T + T A (TV) G:C+TA (TV) T:A+AT (TV) T A + C G (TS) deletion 21 bp TA+C:G (TS) CG-*T:A (TS) G:C+AT (TS) deletion C:G C:G+TA (TS) G:C+AT (TS) deletion C G T A C G (TS) C:G-+TA (TS) G:C+TA (TV) C G - + T A(TS) G:C+AT (TS) deletion C:G A:T+C:G (TV) CG-+A:T (TV) C:G+AT (TV) deletion A:T C:G-+AT (TV) deletion 1046 bp C:G+A:T (TV) deletion 1046 bp C:G+TA (TS) deletion A T C:G+A:T (TS) AT-+T:A (TV) C:G+TA (TS)
Position 1524 1270 1676 1637 1721 1242 1577 1592-1613 1720 1592 1627 1626 1388 1627 1626 1562 1196 1348 1159 1708 1318 1338 1638 1520 1651 664 670-1716 664 670-1716 1399 1397 1329 993 1640
Target Sequence CGAT.TACG AACGACGG TACTAGCAG GGGnACAG GTWGGAC TGTWGTGG TCCACAAAA rn.CGCT CGTCCGGGA CTlTIJ3CTA TI'GCMTA TTTG.GAAT TGGnGCTG ITGC-WTA 'ITTG.GAAT GCTGCGCCG T A T q GAAC AATGPGCA GAAGTAGAA ACAGAGCGG GCTA.CGGC GCGTGACGC CCCWGCGA TGCCAGATG TAAC.GTCT GCTGgTAA ATAA.CAG A GCTGFTAA ATAA.CAGA GAATWTC GGGA.TGAA TGAGAGAAC AAGCIGAAG CACGTGATG
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TABLE 3. DNA Sequence Analysis of Spontaneous LacZ Mutants Isolated from the Liver of Transgenic Mice of Strain 40.6 Mutant 1 2 3 4 5 6 7 8 9
10 12 14
15 16 18 19 20 21 24 25 26 27 28
Sequence Alteration TA-C:G (TS) CG-TA (TS) TA-CG (TS) CG-TA (TS) AT-TA (TV) deletion G:C AT-CG (TV) CG-TA (TS) C:G-T.A (TS) GC+AT (TS) deletion T A deletion ATTA TA-CG (TS) CG+TA (TS) CG+TA (TS) AT-G:C (TS) G:C-AT (TS) CG-T:A (TS) G:C-AT (TS) G:C-AT (TS) TA+C:G (TS) G:C+TA (TV) AT-TA (TV) AT-+G:C(TS) TA-CG (TS) C:G-+T:A(TS) deletion A T
Position 1103 1531 1962 2743 617 1370 1096 1531 461 507 1585 1911/1912 779 2813 1529 634 2142 1196 901 820 2633 1873 1883 1602 1376 1531 2822
Target Sequence CCrCCGCAT CGCGIGCGT GCGACAACG CGA(JTGCCr GATnGGAT CACC.GATG GAGCgCAT CGCGIGCGT AAm-GGCG GCCMGACA AAAA.GGCr GllT..CCG TACCCACGG 'ITC(JTGAGC TACGIGCGC GATGGGCGG CATGATCAG TATnGAAC GAACATCGA GGTCACCAG GAAGCGGCG GACGTAAGC AAAnCCAG CTGGGGAGA AGTGCGATC CGCGTGCGT GAAA.CGGT
mutations are present in a small area of about 500 bp, the 40.6 strain has the mutations spread all over the 3,000-bp lacZ gene. Furthermore, of all transition mutations the target sequence in the 40.6 strain is clearly a methylated CpG-rich region, which is not the case with the 35.5 strain. Therefore, we conclude that the mechanism of mutation induction in the 35.5 strain differs considerably between the two genomic sites. It is well documented that the majority of basepair mutations (68%) appear to be replication dependent.33 It is therefore likely that the high spontaneous mutation frequency in strain 35.5 reflects misincorporations opposite apurinic sites by DNA polymera~es,3~ for example, as a consequence of differences in chromatin structure or the asynchronous replication of active and inactive X chromosomes during cell repli~ation.~~ The latter could be tested by crossing the 35.5 strain with Searle's translocation mice [t(X,16)] and comparing mutation frequency in females where the lambda-gtl0LacZ concatemer is on the normal or on the rearranged X chromosome. If the lambda-gtl0LacZ concatemer is indeed preferentially present on the active X chromosome, this may account for the observed difference in spontaneous mutation frequency between hemi- and homozygote brain D N A (TABLE 1) and would explain the absence of differences in mutation frequencies between hemizygote male and female mice. Our results suggest that the high spontaneous mutation frequency in liver and
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brain DNA of strain 35.5 is due to the location of the lambda-gtl0LacZ concatemer at a highly unstable chromosomal region, making it more susceptible to both germinal and somatic mutagenesis. The large differences in spontaneous mutation frequencies between the 35.5 and the other two strains may therefore indicate the presence of a mutational “hot spot” in the somatic genome that has hitherto only been found for some minisatellite A major question regarding the origin of the high number of spontaneous mutations in strain 35.5 is when these events take place. Indeed, if such high mutation frequencies would be constant, one would expect that at old age virtually all copies of the gene will have undergone a mutational change. We have currently performed only very limited studies on the effect of aging on the mutation rate. Preliminary data of mutant frequencies in liver and brain as a function of chronologic age indicate high interindividual variation, but no dramatic change with age (results not shown). In this regard it is not inconceivable that at some early stage during embryogenesis a burst of mutational activity is responsible for the high mutant frequencies observed. In any case the question as to whether this specific chromosomal site is more susceptible to an age-related increase in mutation frequency remains an intriguing one. Finally, the bacteriophage lambda-based models just described have the major disadvantages that (1) a selection against nonmutant plaques is lacking and (2) expensive packaging extracts are needed. Therefore, a new system has been developed that is based on plasmid rescue by magnetic beads and mutation detection by subsequent transformation of Gal E- Eschen’chiu coli cells.37This avoids the need for expensive in vitro packaging extracts and provides a selection step in which all nonmutants are killed. This system is most suitable for the large-scale studies on spontaneous and induced mutations that are necessary to obtain insight into the relation between somatic mutations and the aging process.
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