Human Molecular Genetics, 2002, Vol. 11, No. 2 191-198
© 2002 Oxford University Press
Somatic expansion behaviour of the (CTG)n repeat in myotonic dystrophy knock-in mice is differentially affected by Msh3 and Msh6 mismatch–repair proteins
Department of Cell Biology, UMC Nijmegen, Nijmegen Center for Molecular Life Sciences, PO Box 9101, 6500 HB Nijmegen, The Netherlands and 1Division of Molecular Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands
Received October 3, 2001; Revised and Accepted November 16, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The mechanism of expansion of the (CTG)n repeat in myotonic dystrophy (DM1) patients and the cause of its pathobiological effects are still largely unknown. Most likely, long repeats exert toxicity at the level of nuclear RNA transport or splicing. Here, we analyse cis- and trans-acting parameters that determine repeat behaviour in novel mouse models for DM1. Our mice carry ‘humanized’ myotonic dystrophy protein kinase (Dmpk) allele(s) with either a (CTG)84 or a (CTG)11 repeat, inserted at the correct position into the endogenous DM locus. Unlike in the human situation, the (CTG)84 repeat in the syntenic mouse environment was relatively stable during intergenerational segregation. However, somatic tissues showed substantial repeat expansions which were progressive upon aging and prominent in kidney, and in stomach and small intestine, where it was cell-type restricted. Other tissues examined showed only marginal size changes. The (CTG)11 allele was completely stable, as anticipated. Introducing the (CTG)84 allele into an Msh3-deficient background completely blocked the somatic repeat instability. In contrast, Msh6 deficiency resulted in a significant increase in the frequency of somatic expansions. Competition of Msh3 and Msh6 for binding to Msh2 in functional complexes with different DNA mismatch-recognition specificity may explain why the somatic (CTG)n expansion rate is differentially affected by ablation of Msh3 and Msh6.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Myotonic dystrophy [MIM 160900; dystrophia myotonica 1 (DM1)] is the most prevalent form of adult muscular dystrophy, with multisystemic features and a highly variable clinical phenotype (1). Careful clinical evaluation and genetic testing of large groups of patients with this dominantly inherited disorder has revealed that at least two forms of disease and underlying genetic causes can be distinguished (2–8). The genetic basis for the most common form of DM, DM1, is an expanded CTG repeat in the 3'-untranslated region (UTR) of the DM protein kinase (DMPK) gene (9–12) and in the promoter region of the immediately adjacent homeodomain SIX5 gene (3) on chromosome 19q13. Myotonic dystrophy type 2 [MIM 602668; Dystrophia Myotonica 2 (DM2)] displays a remarkable identical phenotype (13) and is recently linked to a CCTG expansion in the first intron of the zinc finger protein 9 gene (ZNF9) on chromosome 3q21 (14).
Because of the striking parallels between these mutations, both of which eventually give rise to large microsatellite expansions in non-coding areas of muscle-expressed (pre)mRNAs, it has been speculated that the expansions create a distinct type of dominant gain-of-function mutation (6,8,15). In one proposed mechanism the mutations would have cis-effects on processing, routing or translation of the DMPK or ZNF9 mRNAs proper. This mechanism assumes that DMPK and ZNF9 work in the same cellular pathway and the same set of cellular functions may be distorted. More likely is that the mutations cause the same type of trans-acting toxic effects on nuclear metabolism by sequestering specific proteins that are required for pre-mRNA splicing or export (16,17). Hypotheses that postulated how expanded repeats could alter expression of neighbouring genes by position effect variegation have lost attractiveness. Since there is no similarity between the DM1 and DM2 gene clusters it is difficult to see how deregulation of expression of completely different subsets of genes would produce such a remarkable similarity in disease phenotype.
In DM1 it is particularly well documented that the size of the repeat is correlated negatively with age of onset and positively with the severity of the disease phenotype. As repeats of >40 CTG will expand further in successive generations, and large repeats are very unstable in the germline and heavily biased towards further expansion, this provides the molecular explanation for the ‘anticipation’ phenomenon. In the literature there is a vast amount of data indicating that somatic mosaicism in DM1 is also very high. Small-pool PCR experiments have demonstrated that the CTG repeat in human tissues displays an age-dependent (18), expansion-biased (19), tissue-specific (20,21) length heterogeneity that appears to be mediated via multiple successive small length change events [mostly size increase (22)], starting early after gestation. In addition, transgenic animals that carry the DM CTG repeats show both intergenerational and somatic instability. Strikingly, a strong tissue-type dependence of repeat length instability has been observed, suggesting that tissue-specific factors may be involved (23,24).
Unfortunately, not much is known about the molecular basis underlying the repeat expansion, or the mechanisms involved in creating the tissue variability in CTG length. Recent studies on the expansion mechanism of the CAG repeat in Huntington disease (HD) in transgenic mice (25–27) pointed to the possible involvement of components of the DNA repair machinery. The data obtained support models in which expansion arises by single-stranded DNA gap repair and is not dependent on mitotic replication or recombination between homologous or sister chromatids.
Here we report on a study of a new knock-in mouse model with a long CTG84 repeat placed into the mouse DM1 locus, at a site exactly homologous to that where it is found in human patients. This enabled us to examine the (cis-acting) effects of proximal gene regions and (trans-acting) factors that relate to tissue type and ageing. Importantly, in order to clarify the role of individual key proteins in the DNA mismatch and repair process we crossed a CTG97-DM1 locus into backgrounds with Msh3 and Msh6 deficiency. Our results demonstrate that, although Msh3 and Msh6 both form functional complexes with the Msh2 repair protein (28), they have a profoundly different role in the recognition and/or processing of anomalous DNA structures that may form in or around long CTG tracts at the DM1 locus.
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RESULTS |
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Production of a DM1 CTG knock-in mouse model
To generate a mouse model with an expanded human (CTG)84 repeat in the cognate position of the endogenous Dmpk gene, we used a multistep replacement mutagenesis procedure with targeted recombination-insertion and Cre-loxP based excision steps (29) in mouse 129/Ola-derived ES cells (Fig. 1). As a control for monitoring the effect(s) of the replacement of exons 13–15 by homologous human sequences, we created mutant mice with a non-expanded ‘normal’ (CTG)11 repeat. Several independent ES cell clones were obtained from which highly chimeric (CTG)84- or (CTG)11-Dmpk allele transmitting males were derived. All heterozygous and homozygous mutant mice obtained during subsequent breeding were indistinguishable from their normal littermates, fertile, and displayed no overt outward anomalies. Northern and western blot analyses revealed that the mutant mice expressed the predicted chimeric mouse–human Dmpk transcripts, resulting from alternative splicing (12,30) of mouse exon 12 to either human exon 14 or 15, albeit at reduced levels. However, their tissue distribution profile was exactly similar to that observed for endogenous Dmpk mRNA and protein isoforms (12), indicating that the chimeric allele obeys the same gene-regulatory control as the wild-type allele (data not shown; D.G.Wansink, M.M.Coerwinkel, W.J.A.A.van den Broek, P.J.T.A.Groenen and B.Wieringa, manuscript in preparation).
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Intergenerational segregation of the CTG repeat
We next tested how the (CTG)84 allele behaves upon intergenerational transmission. In mice from cohorts with the mixed C57Bl/6 x 129/Ola background, the repeat was essentially stable and only (CTG)84,85,86 alleles were found. However, during breeding efforts to place the (CTG)84 allele in a pure C3H background we discovered one lineage of mice with slow but progressive increments in repeat size (Fig. 2A). The average repeat length in tail DNAs in this pedigree increased from 84 to 97 CTGs in three generational jumps and is now at 100 in the F7 generation. One major contraction was detected (–16 CTGs). In the cohorts analysed, similar numbers of contractions and expansions were seen: CTG repeat expansions (+1 to +8 triplets) in 42 out of 132 parent–sibling pairs, contractions (1–2 triplets, with the exception mentioned above) in 38 out of 132 parent–sibling pairs. This means that the frequency of mutation is different from the situation in human DM families, where even in non-manifesting carriers—with (CTG)41–95 repeats—the incidence of significant repeat-length alterations is in the order of
90–95% [mainly expansions (31)]. Sequence analysis of genomic tail DNA of (CTG)84 mice revealed that the repeats were without imperfections, which theoretically could hamper repeat instability.
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Cell-type-dependent factors are involved in CTG-repeat expansion
To study repeat behaviour at the somatic level and monitor the effects of genetic background we measured (CTG)84 allele sizes in various tissues from several heterozygous transgenics (successive generations, 3–22 months of age) held on a mixed C57BL/6 x 129/Ola background or crossed once into a Balb/C, FVB or C3H environment. Repeat patterns obtained after PCR amplification consistently displayed size increments in all tissues when compared to the pattern generated from the plasmid-borne (CTG)84 repeat (Figs 2B and 3A). When tissues of animals with different backgrounds were compared (n = 2, randomly selected animals from each group with mixed Balb/C, FVB, 129 or C3H background; analysis at both 6 and 12 months of age) those with a C3H background contribution
50% showed the highest fractional contribution of expanded alleles (Fig. 3A).
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Strikingly, there was pronounced instability in DNA from stomach, kidney and small intestine, in all animals tested. The instability did not differ between male and female animals and appeared progressive with age (Figs 2B and 3B). This indicates that instability persists throughout adult life as seen in DM patients (19,32) and involves an increasing number of cells in a given tissue. Based on these observations we assume that genetic constitution is important for the frequency of expansion events, influencing both the length of individual repeats and the number of cells involved as a function of time.
When tracked as a function of cell type, repeat expansion was mainly seen in cells from the mucosa and sub-mucosa fraction and much less in cells from the muscularis externa fraction of the stomach [i.e. in which Dmpk is predominantly expressed (12)] (Fig. 3B). Also, in cells scraped from the luminal side of the small intestine repeat expansion was obvious. Interestingly, no mosaic length distribution with large expansions was seen in other mixed cell preparations with actively proliferating (stem) cells like sperm, bone marrow, blood or skin (Fig. 2B). This suggests that expansion is not, or not consistently, related to cell-cycle progression per se and may involve non-mitotic events. We are currently analysing different cell types of kidney, lung and bladder to explore the possibility that a high frequency of repeat expansion events might be a consistent feature of (epithelial?) cell types present in areas that connect with the outside of the body, i.e. in the alimentary, respiratory or genitourinary tracts.
Guarding role of Msh3 and Msh6 repair proteins
The somatic mutation frequency and clear ageing effects in stomach, kidney and intestine of our (CTG)84 mice offered us a unique context for searching specific trans-acting factors that could stimulate or suppress instability, or promote transition to full (CTG)n repeat instability, as seen in humans. Previous studies have indicated that MMR protein Msh2 may have a crucial role in repeat expansions (25,26,33). To obtain insight in the significance of other repair complex proteins we placed a (CTG)97-Dmpk allele, obtained from the pedigree shown in Figure 2, into an Msh3–/– or Msh6–/– background (34). Multiple comparisons of repeat size distribution between tissues from DM-transgenic Msh6–/– (CTG)97+/– and Msh6+/– (CTG)97+/– littermates (n = 3) at 6 and 12 months of age showed a consistently greater repeat instability in animals that lack Msh6. Although the distribution over tissues with high, intermediate and low expansion frequencies remained qualitatively similar, the stomach, kidney and also liver now showed a significantly stronger culmination of expansion events (Fig. 3C). It may be of interest here that we have found alleles with almost double-sized repeat lengths in an isolated liver tumour that had developed in one of our 12-month-old Msh6–/– (CTG)97+/– mice (data not shown). Repeat length profiles in other tissues, like heart, muscle, brain, tongue, tail and sperm, were similar to those in wild-type (CTG)97+/– animals.
In contrast, the (CTG)97-Dmpk allele placed in an Msh3–/– background yielded profiles similar to that of the CTG84-control plasmid, pointing to a complete absence of somatic CTG-repeat instability (Fig. 3D). Furthermore, Msh3 deficiency also completely protected against age-related (at least up to 12 months) instability.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In this study we have analysed the instability of a perfect (CTG)84 repeat (cloned from the DMPK locus of a DM family) in a new ‘second generation’ mouse model. When placed in the cognate chromosomal environment of the mouse genome, i.e. the 3'-UTR of the Dmpk gene on the proximal arm of mouse chromosome 7, repeat length behaviour was grossly reminiscent of that in somatic tissues of human patients and strikingly, also of that of randomly inserted (CTG)50–500 repeats in soma of different transgenic mouse lineages (17,23,24,35,36). Furthermore, the (CTG)84 repeat was only marginally unstable during intergenerational transmission. This is unlike the human situation, where in 90–95% of transmissions from a non-manifesting carrier to offspring a length change (predominantly expansion) would be expected for repeats in the size range between 41 and 95 triplets (31). Similar semi-stable intergenerational expansion behaviour has been reported for the transgenic DM mouse models with randomly positioned inserts and different segments of human flanking DNA. Also, for mouse strains carrying trinucleotide-repeat segments from the human HD, DRPLA or fra-X genes (37–39) repeat length changes were relatively modest and appeared less frequent than in the human situation. Taken together, these observations seem to exclude a species-specific and dominant role for flanking sequences. Other factors that may determine intergenerational repeat expandability may relate to species-specific parameters involved in setting the reproductive life-span (40), the number of cell divisions during gametogenesis (32,38) or the efficiency of DNA metabolism and repair in the germline compartment (27,33). Interestingly, Kovtun and McMurray (27) reported that expansion in a HD mouse model is limited to post-meiotic haploid cells and most likely involves gap repair by Msh2 containing complexes. Future follow-up studies in which well defined trinucleotide repeats can serve as genomic reporter elements should therefore be directed to inter-species and inter-individual comparison of mechanistic steps in this process.
Some of the steps involved in length mutation of trinucleotide repeats may be shared between cells in the germ-line and somatic tissues. Of particular interest is the observation that our (CTG)84 knock-in model exhibited a significant and age-dependent somatic instability in all tissues examined, but most pronounced in stomach, kidney and small intestine. Closer inspection of specific cell types of both stomach and small intestine revealed that repeat expansion was mainly present in the (sub) mucosa fraction, with a high fractional content of actively dividing cells. However, such simple correlation between expandability and cell division or genome replication rate did not exist for kidney (a tissue with relatively few dividing cells and the highest rate of expandability), bone marrow (high content of actively proliferating cells, but low expandability) or brain (very low number of dividing cells, moderate instability) (Fig. 2B). This confirms other studies [including those using cells with repeats in tissue culture (41)] and suggests that tissue-specific factor(s) rather than the frequency of replication per se controls the rate of somatic repeat expansion. Of particular interest seems the finding that the most conspicuous instability in our model and in other DM-transgenic lines was observed in the kidney. In cells present in the lining of the gastro-intestinal tract the repeat was also very unstable, but a comparison to tissues in other studies is not possible, because they were not analysed. Merely as a suggestion, we would like to point out that the kidney and inner intestinal layers are particularly rich in epithelial cells. These cells may perhaps need highly active DNA repair, because they are constantly in direct contact with the variable external milieu (sometimes containing genotoxic waste products) in the body. Further study and comparison of tissues like lung or bladder, and the DNA repair activity therein, is necessary to see if this speculative correlation holds.
Involvement of proteins from the DNA repair and recombination machinery is a salient feature of many studies that have pointed to the importance of post-mitotic events in trinucleotide instability in patients and cellular and animal model systems. One mechanism implicated is the activity of the mismatch–repair (MMR) machinery. For example, there is solid evidence that a core component in the MMR complex, Msh2, has affinity for DNA structures formed by CTG repeats (25). Most direct evidence comes from the group of Manley et al. (26) who have shown that absence of Msh2 has a stabilizing effect on the HD trinucleotide repeat. Here we report that Msh3 deficiency mimics this effect of Msh2 deficiency, in that somatic repeat instability of the (CTG)84 repeat in our knock-in model is completely absent. In contrast, in an Msh6 deficient background higher instability was seen; particularly in stomach and kidney. Our finding that Msh3 and Msh6 have opposed effects on trinucleotide repeat instability is an unexpected result. For mismatch recognition, MSH2 forms heterodimers with either MSH6 or MSH3, depending on the type of mutation that needs to be repaired (42,43). It is generally thought that the MSH2/MSH6 complex functions in base:base mispairs whereas the MSH2/MSH3 complex is primarily responsible for the recognition of larger insertion/deletion loops. However, there is functional redundancy, as both Msh2–Msh3 and Msh2–Msh6 repair complexes recognize single base insertion/deletion mispairs (43). The (CTG.CAG)97 DNA may be entangled in either slipped-strand structures or misaligned recombination products between sister chromatids (44,45). Our data suggests that recognition and stabilization of such large anomalous structures by Msh2/Msh3 heterodimers promotes misincorporation of additional (CTG) triplets. Over age, this could lead to involvement of an increasing number of cells, each of which could undergo multiple rounds of misincorporation, resulting in gains of relatively large tracts of CTGs. As the stability of individual Msh3, Msh6 or Msh2 proteins depends on their opportunity to heterodimerize (34) the level of the Msh2/Msh3 complex in Msh6-deficient cells could even exceed that in wild-type cells, leading to increased instability. In contrast, the presence of only the Msh2/Msh6 complex in Msh3–/– mice may help to protect against somatic expansion. Unfortunately, the precise mode of action of this complex in the process of recognition, untangling, or gap-repair of anomalous structures in trinucleotide repeats is currently not precisely known. The mechanistic aspects of repeat (de)stabilization in our repair-deficient model therefore remain enigmatic.
In summary, our data suggest that species context, cell-type and genetic background parameters work together with the MMR machinery in determining somatic (CTG)n repeat instability in the DM locus in mice. Whether this picture also holds for the somatic and meiotic instability of the repeat in DM patients remains to be seen.
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MATERIALS AND METHODS |
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Construction of the targeting vector
The targeting vector was derived from cosmid mDMRc8 (mouse 129/Sv specific) (14) and contains the 5.3 kb EcoRV–StyI fragment (spanning nucleotide positions 4873–10200 of the genomic mDMPK sequence) as the upstream segment, and the 2.7 kb NsiI–EcoRI fragment (spanning nucleotide position 11865 in the Dmpk gene to nucleotide position 2347 in the Dmahp gene) (5) as the downstream segment of homology. Between these homologous arms, bracketed by a blunted StyI site at position 10200 and the natural NsiI site at position 11865 of the mouse sequence, we placed a loxP flanked Neo/HSV-tk cassette (courtesy of Dr P.Krimpenfort, The Netherlands Cancer Institute, Amsterdam, The Netherlands) and a human segment containing the DMPK exon 13–15 area. One type of human fragment used carried a normal (CTG)11 repeat, the other contained an expanded (CTG)84 repeat, and these were obtained by PCR on DNA from cosmid F100263 [=3C7 (46)] and whole genomic blood DNA from a non-manifesting DM carrier, respectively. Fragments were tailored in several cloning steps, during which the mouse StyI (intron 12)–BamHI (position 10379, exon 13, including the splice acceptor site) was removed. As a result, the endogenous mouse exon 13–15 gene segment [between BamHI and NsiI (position 11865)] was exchanged for a human segment between the BamHI at position 12129 and an artificially introduced NsiI site at position 13655 (corresponding to the naturally occurring NsiI at position 11865 in mDMPK exon 15) carrying an expanded (CTG)84 or non-expanded (CTG)11 repeat.
Targeted ‘humanization’ of the 3' part of the DMPK locus in ES cells and generation of mouse models
E14 ES cells, a kind gift of M.L.Hooper, were maintained as described by Bradley (47) and van Deursen and Wieringa (48) and 107 ES cells were mixed in medium with XbaI (Dmpk gene position 5235) linearized targeting vector DNA (12 µg) and electroporated at 250 V and 500 µF using a Gene Pulser (Bio-Rad) apparatus. Cells were seeded onto SNLH9 feeder cells and positive selection was started after 24 h with 300 µg/ml G418. Cell colonies were picked after 8–10 days of selection and screened by Southern blot analysis after BglII digestion of genomic DNA using 5' (BglII–BamHI) and 3' (EcoRI–BglII) probes that flank the sequences present in the targeting vector (Fig. 1A and B). Independent homologous recombinants with normal karyotypes were used for Cre-mediated excision of the Neo/HSV-tk cassette from intron 12, leaving only the loxP sequence (plus polylinker sequences from the vector) as a chromosomal mark. To this end, clones were transfected with a CMV promoter driven Cre-recombinase expression plasmid and selection with FIAU (0.2 µM, a kind gift of Bristol Myers) starting at day 6 after electroporation. Cell colonies were picked at day 10 of selection. Successful excision of cassette sequences was monitored by Southern blot analyses of HindIII-digested ES DNA and hybridization with a genomic HindIII–StyI probe (Fig. 1A and B).
Clones that had undergone the correct genomic change(s) were identified at >95% frequency for the (CTG)84 allele and at <10% frequency for the (CTG)11 allele and were karyotyped again. Ultimately, ES cells from independently obtained (CTG)84 and (CTG)11 clones were microinjected into C57BL/6 blastocysts and used to generate chimeric mutant offspring (47). Transmitting males were used in further breeding programs. All procedures involving mice were approved by the Animal Care Committee of the University of Nijmegen and conformed to the Dutch Council for Animal Care and NIH guidelines.
Genotyping and analysis of instability of the (CTG)n repeat
Msh3 and Msh6 heterozygous knock-out male mice on a 129/Ola background were crossed with heterozygous Dmpk (CTG)97 repeat female mice [on a mixed C3H (94%)-129/Ola/C57BL/6 (6%) background]. F1 (50% C3H/50% 129/Ola) generation mice were intercrossed to obtain mice homozygous or heterozygous for Msh3 or Msh6 knock-out alleles and heterozygous for the CTG97 allele. Littermates with the mixed inbred background were analysed for repeat instability as described below. Msh3 or Msh6 genotypes were determined by PCR (34). DNA was prepared from tail biopsies or tissues samples according to standard procedures. For certain experiments stomach and small intestine were first fractionated into a mucosa-enriched fraction and a (smooth) muscle-enriched fraction. Briefly, stomach and small intestine were cut open and rinsed in PBS. Using a scalpel, the mucosa fraction (mainly the tunica mucosa and the tela submucosa) was scraped off, leaving the muscular fraction (mainly the tunica muscularis, tela subserosa and tunica serosa) behind and both preparations were analysed separately. For routine typing, PCR was performed with the forward primer 226 (5'-GAAGGGTCCTTGTAGCCGGGAA-3', matching the DMPK gene sequence between nucleotide positions 13206 and 13227), and the reverse primer 225 (5'-GGAGGATGGAACACGGACGG-3', matching nucleotides 13353–13372). The position of the (CTG)11 repeat is assigned nucleotide positions 13230–13262. Genomic DNA (100 ng) was PCR amplified in a mixture containing 100 ng of each primer by a standard protocol (34 cycles at 94°C for 1 min, 58°C for 1 min and 72°C for 2 min) and amplified products were electrophoretically resolved on agarose gels. The PCR yields a 166 bp band for the humanized (CTG)11 allele and a 385 bp band for the humanized (CTG)84 allele as originally cloned. For more precise repeat length estimates PCR was performed with primer 226 and a Cy-5 fluorescently labelled primer 225. Amplified products were mixed with formamide loading buffer, heated for 10 min at 96°C and subjected to electrophoresis on 6% denaturing acrylamide–urea gels in the ALF DNA sequencing system (Amersham Pharmacia Biotech, Uppsala, Sweden). To determine the number of (CTG) repeats, the highest peak in the trace of the (CTG)84 plasmid was used as a reference point. Somatic instability was analysed by comparing peak area distributions in repeat profiles. Intergenerational instability was studied by comparing the lengths of the main peak(s) in profiles of individual tail DNA products. For repeat sequence verification genomic DNA samples were PCR amplified using the forward primer 14 (5'-GCCAATGACGAGTTCGGA-3', matching nucleotides 12674–12691) and the reverse primer 227 (5'-TTTGCACTTTGCGAACCAAC-3', matching nucleotides 13390–13409). The sequencing reaction was performed with the fluorescently labelled forward primer DM-A [5'-CAGTTCACAACCGCTCCGAGC-3' (32), matching nucleotide positions 13091–13111] and DNA sequence determined on the ALF system.
Accession numbers
Numbering of sites is according to GenBank database accession nos L08835 and Z38015 for the human and mouse DMPK genes, respectively.
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ACKNOWLEDGEMENTS |
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We thank Dr P.Krimpenfort, Netherlands Cancer Institute, Amsterdam for help and advice with the Cre/loxP experiments. This study was supported by grants from the American Muscular Dystrophy Association (MDA), the Association Française contre Les Myopathies (AFM), the Prinses Beatrix Fonds (grant 98-0102) and the Dutch Cancer Society (grants NKI 2000-2233 and KUN 99-1915).
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +31 24 3614329; Fax: +31 24 3615317; Email: b.wieringa@ncmls.kun.nlPresent address:Patricia J. T. A. Groenen, Department of Pathology, UMC Nijmegen, Nijmegen, The Netherlands
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REFERENCES |
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