Human Molecular Genetics, 2000, Vol. 9, No. 8 1185-1194
© 2000 Oxford University Press
Transgenic mice carrying large human genomic sequences with expanded CTG repeat mimic closely the DM CTG repeat intergenerational and somatic instability
INSERM UR383, Hôpital Necker-Enfants Malades, Université René Descartes Paris V, Paris, France, 1Division of Cardiology, Department of Medicine, Baylor College of Medicine, Houston, TX, USA and 2Laboratoire de Biochimie, Hôpital Ambroise Paré, Boulogne, France
Received 7 January 2000; Revised and Accepted 29 February 2000.
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ABSTRACT |
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Myotonic dystrophy (DM) is caused by a CTG repeat expansion in the 3'UTR of the DM protein kinase (DMPK) gene. A very high level of instability is observed through successive generations and the size of the repeat is generally correlated with the severity of the disease and with age at onset. Furthermore, tissues from DM patients exhibit somatic mosaicism that increases with age. We generated transgenic mice carrying large human genomic sequences with 20, 55 or >300 CTG, cloned from patients from the same affected DM family. Using large human flanking sequences and a large amplification, we demonstrate that the intergenerational CTG repeat instability is reproduced in mice, with a strong bias towards expansions and with the same sex- and size-dependent characteristics as in humans. Moreover, a high level of instability, increasing with age, can be observed in tissues and in sperm. Although we did not observe dramatic expansions (or ‘big jumps’ over several hundred CTG repeats) as in congenital forms of DM, our model carrying >300 CTG is the first to show instability so close to the human DM situation. Our three models carrying different sizes of CTG repeat provide insight on the different factors modulating the CTG repeat instability.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONResultsDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Myotonic dystrophy (DM) results from the expansion of a CTG repeat located in the 3'UTR of the DM protein kinase (DMPK) gene (1–4). The triplet repeat is also located in a large CpG island encompassing the gene encoding the DM-associated homeobox protein (DMAHP), which is certainly involved with DMPK in the pathogenesis of DM (5). In the normal population, the repeat varies between 5 and 37 CTG and remains stable (2–4,6). Repeats of >40 CTG expand further in successive generations and generally, the size of the repeat is correlated negatively with age at onset and positively with the severity of the clinical phenotype (7–10). The discovery of trinucleotide ‘dynamic’ mutations, associated with a growing number of inherited human disorders, has provided the molecular explanation for the ‘anticipation’ phenomenon (7), particularly evident in DM.
Analyses of DM family pedigrees have shown that >90% of transmissions result in the expansion of the repeats, whereas only 6–7% result in contraction (11,12). However, the dynamics of the CTG repeat instability associated with DM depend on factors such as the size of the repeat and the sex of the transmitting parent. When the CTG repeats are 
100 units, amplifications are much larger after paternal transmissions, accounting for previous observations of an excess of males in the earliest generations of DM pedigrees (8,11,13,14). A similar bias is observed for CAG repeats in CAG repeat-associated neurological diseases (15). The dynamics of the triplet repeat are more complex in DM. For repeats of 
500 units, there is a negative correlation between the size of the repeat in the transmitting father and the intergenerational variations in DM families. The CTG repeat tends to contract only after paternal transmission, whereas very large amplifications are observed following maternal transmission. The inheritance of very large repeats in maternal transmission and the apparent selection against repeats of >1000 CTG in sperm result in severe congenital cases of DM being selectively of maternal origin (16). In DM patients, differences in the repeat length have been observed between tissues, and the expanded allele is detected as a broad smear in Southern blot and PCR analyses (10,13,17–20). Small-pool PCR experiments have demonstrated a strong bias towards expansion in blood and showed that somatic heterogeneity is size dependent and increases with age (21–23). These observations reflect inter- and intra-tissue somatic instabilities and suggest that tissue-specific factor(s) control the rate of expansion. The somatic instability observed in most trinucleotide repeat-associated diseases is particularly strong in DM. Instability appears very early in embryogenesis, seems to increase after 16 weeks of gestation and then continues into adulthood, probably by the accumulation over time of small mutational changes (18,24). Variation in the number of CTG repeats is also very evident in the sperm of DM patients, with a tendency towards an increase in size. However, alleles containing >1000 repeats have not been detected by small-pool PCR. This is consistent with genetic anticipation and with a size limitation for paternally inherited amplifications. It has been suggested that the sex-specific intergenerational instability may result from differences in the number of mitotic divisions during spermatogenesis and oogenesis (19) and from the selection of allele >1000 repeats during spermatogenesis. However, we cannot exclude the possibility that intergenerational amplifications result from both germline and postzygotic expansion.
The length and purity of the array are determinant factors for trinucleotide repeat instability. However, mutation rates, size variation and the tendency towards expansion or contraction seem to differ between the various disease loci, emphasizing a possible influence of the sequences flanking the repeat on instability processes (25). Bacteria and yeast models have provided evidence that the stability of CAG/CTG repeats also depends on their orientation with respect to the origin of replication, and on DNA mismatch repair system efficiency (26–32). The formation of abnormal structures, such as hairpins and slipped-strand DNA, at the repeat arrays has been demonstrated in vitro and is strongly suspected to be involved in instability (33,34). Although the situation may be different in humans, experimental systems such as in vitro studies, bacteria, yeast and cultured cells have already provided some clues with regard to the unresolved instability mechanisms. Nevertheless, trinucleotide repeat instability must be reproduced in animals to enable us to study, in vivo, the mechanisms involved during gametogenesis and development and to explain the size- and sex-dependent dynamics observed in affected families.
We and others have reproduced moderate CTG/CAG repeat instability in mice and have demonstrated that, although the first attempts were unsuccessful, mice are a suitable model for reproducing the instability of triplet repeats and elucidating the mechanisms involved (35–46). In mouse models, instability was achieved either with large repeats or with an appropriate genomic context. This emphasizes the importance not only of repeat size, but also of the genomic flanking sequences. Transgenic mice models have also provided some evidence that several different mechanisms (replication dependent, replication non-dependent and transcription dependent) might be involved in instability (41,42,47). However, there are differences between human and mouse models in that rates of instability are lower in mice and there are tendencies towards contraction of the repeats with maternal inheritance. We studied the CTG repeat instability involved in DM by producing transgenic mice carrying very large human genomic sequences (45 kb) and large CTG repeats (>300 CTG), cloned from individuals from the same family who exhibit dramatic intergenerational changes. We compared these mice with mice carrying the same genomic context of 45 kb but with a normal repeat (20 CTG) or a small amplification (55 CTG), and studied intergenerational and somatic instability. We found that transgenic mice carrying large human genomic sequences surrounding a large repeat reproduced all the CTG repeat instability characteristics of DM except for very large amplifications or ‘big jumps’. Comparison of our model with previous models provides insight into the various factors regulating trinucleotide repeat instability mechanisms.
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Results |
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Cloning of 45 kb DM genomic fragments
The various fragments used to generate the transgenic mice were cloned from human genomic DNA extracted from lymphoblastoid cells. Two cell lines were derived from two patients of the same family. We chose a mildly affected patient (R.D.) with 50–70 CTG repeats and his affected daughter (N.G.) carrying an expanded allele inherited from the father, with ~500 CTG. She gave birth to two affected children with the congenital form of the disease and large expansions > 2000 CTG. We made two cosmid libraries with the SCos1 modified vector ‘Scossbs’. We screened these libraries with the DMHR4/5 probe spanning exon 2, intron 2 and exon 3 of the DMPK gene (Fig. 1). We first obtained clone DM55 carrying ~45 kb of human genomic DNA sequence as described in Gourdon et al. (39). It carries the DMPK gene, the DMWD gene (48) and the DMAHP gene (5) (Fig. 1). From the second library, we isolated a clone with ~360 CTG (DM300) from the expanded allele and a clone with 20 CTG (DM20) from the normal allele. To obtain a fragment identical in all but the size of the repeat, we replaced in the DM55 clone the NdeI–ClaI fragment carrying the DMPK gene from exon 2 and the DMAHP gene by the corresponding fragments recovered from DM300 and DM20 (Fig. 1). The DM55 and modified DM300 clones were identical except that they carried CTG repeats of different sizes. The NdeI–ClaI fragment with 20 CTG originated from a normal allele and did not contain the Alu repeat insertion localized in DMPK intron 8 of the mutated alleles.
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Production of transgenic mice containing the DM region with CTG repeats of various lengths
The 45 kb insert was injected into B6D2/F1 mouse embryos as previously described (39) and, starting with the second generation, transgenic animals were crossed with C57BL/6 mice. We obtained two lines with 20 CTG (DM20-949, DM20-954), seven lines with 55 CTG [described in (39)] and three lines with at least 300 CTG (DM300-328, DM300-1112, DM300-1177). BamHI digests of genomic DNA from some transgenic offspring of each line were probed with ExtC 7.1 (Fig. 1) to map the 5' end of the constructs in terms of junction fragments and tandem repeat bands. We were unable to map the 3' end of the constructs accurately because probes corresponding to this end, which contains highly repeated sequences, gave uninterpretable results. The copy number of the integrated fragment in each of the seven lines was determined by Southern blot hybridization with probe P59 (Fig. 1) and comparison of the density of the signal obtained with that for a fragment from an endogenous single copy gene and known standards. Cosmid DM55 was also used as a probe on Southern blots, to check the integrity of the transgene in each line. The size of the CTG repeat in each founder was determined by PCR using primer 101/102 (see Materials and Methods) surrounding the repeat, and separation of the products on polyacrylamide gels. For the DM300 lines, the number of CTG repeats in each PCR product was deduced by comparison with a 100 bp DNA ladder run on the same gel. The results are summarized in Table 1.
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Size threshold for CTG repeat instability in mice
We assessed the size of the CTG repeat in each line by PCR using tail DNA extracted at 4 weeks of age. As expected, in the two lines carrying the normal 20 repeat, we observed no change in the number of CTG repeats in ~180 transgenic mice from five successive generations (data not shown). In six of the seven lines carrying the small 55 CTG mutation, we have already reported moderate instability with a majority of increases ranging between +1 and +6 CTG in 7% of the transgenic descendants and decreases in < 1% (39).
In the lines carrying at least 300 CTG, 215 transgenic descendants were analyzed for the DM300-328 line, 25 for the DM300-1112 line and 22 for the DM300-1177 line. We observed that intergenerational instability and CTG size variations were dramatically increased in comparison with the DM55 lines. In the descendants, we observed expansion in 86.5%, contraction in 5% and no change in 8.5% for line DM300-328; 88% expansion, 2% contraction and 4% no change for the large CTG repeat in line DM300-1112; and 95.5% expansion and 4.5% contraction for line DM300-1177. Strikingly, this proportion of expansions and contractions of the CTG repeats is similar to that observed in DM families [expansion in >90% and contraction in 7% (11,12)]. The distribution of the size variations in each line is shown in Figure 2. In line DM300-328, which was studied more extensively, most changes were expansions of +1 to +60 CTG in one generation. So far, we have found expansions of 3–10 CTG in line DM300-1112 and of 2–42 CTG in line DM300-1177. Interestingly, when we compared the CTG repeat variations in the three integrated fragments carried by line DM300-1112, we observed that the three CTG repeat arrays expanded or contracted independently of each other in successive generations (data not shown).
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The plot showing size variations in line DM300-328 (Fig. 2) demonstrates a significant difference between paternal and maternal transmission (P < 0.0001), with greater amplification after paternal transmission (by a mean of +20 CTG) than after maternal transmission (mean of +9 CTG). Considering only the descendants, no significant difference in CTG repeat size variations could be observed between male and female offspring. The bias observed after paternal and maternal transmissions in line DM300-328 was detectable in line DM300-1177 but not in line DM300-1112. In DM300-1112, the integration site or the complex structure of the three copies integrated in tandem repeat may affect the extent to which the CTG repeat is amplified.
These results demonstrate that, as in human DM patients, there is a threshold of instability between 20 and 55 CTG in transgenic mice, and that intergenerational instability increases with the size of the inherited repeat and with a strong bias towards expansion. Retaining the human flanking sequences of the CTG repeats enabled us to reproduce in mice the same bias towards expansion observed in humans. In addition, larger amplifications were obtained after paternal transmission. The observation that different CTG repeat arrays increased independently after transmission in DM1112 individuals suggests that intergenerational instability mechanisms act randomly on the CTG repeat but obey probability laws in terms of the size of the repeat and the sex of the transmitting parent.
Influence of CTG repeat length and sex of the parent
We studied the effect of the sex of the transmitting parent in more detail by plotting CTG repeat size in the offspring (Fig. 3A) and changes in the length of the CTG repeat (Fig. 3B) against the size of the CTG repeat in parents for DM300-328 transgenic mice (215 animals). As in human DM families, the influence of sex seemed to be size dependent (Fig. 3A). Linear regression analysis suggested that, for repeats of less than ~370 CTG, amplifications in the next generation were larger if transmitted by a male than if transmitted by a female. However, this difference between male and female transmission may not exist for larger repeats. This is strengthened by the tendency towards smaller amplifications when the size of the transmitting male increased (see the negative correlation between the size variations in the offspring and the CTG repeat length in the transmitting male parent only; Fig. 3B; r = –0.321; P < 0.0001).
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Influence of age
We investigated whether the age of the transmitting parent affects CTG repeat amplification after male and female transmission by analyzing the 215 transgenic descendants in line DM300-328 according to the sex and age of the transmitting parent at the birth of the offspring (Fig. 4). We plotted the size of the expansion in descendants against the age of their transgenic parent and found, by regression analysis, that there was a positive correlation (r = 0.574; P < 0.0001) for paternal but not for maternal transmissions. The same positive correlation was observed when we plotted the sizes of expansions after transmission from males of the same size, aged only between 10 and 40 weeks, or from separate males that we bred continuously. Thus, in our transgenic model carrying >300 CTG, expansions increased in size with the age of the transmitting male, but not with the age of the transmitting female. Multiple regression analyses simultaneously taking into account parental CTG repeat sizes and the age of the transmitting parent showed the same significant correlations between sex, CTG repeat size or age of the parents and variations in CTG repeat size in the offspring (data not shown).
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Somatic instability is size- and age-dependent
In the two lines carrying the normal repeat of 20 CTG, we analyzed CTG repeats in several tissues from young and old mice. We detected no change in CTG repeat length, showing that the normal 20 CTG repeat is somatically stable (data not shown). In the line carrying the small expansion (55 CTG repeats), we have previously reported inter- and intra-tissue somatic instabilities with increases of up to +12 CTG in the tissues presenting the largest somatic mosaicism (liver, pancreas and kidney). Mosaicism increased with age and all the mice from the various DM55 lines analyzed showed the same pattern of somatic mosaicism, suggesting that integration sites do not affect somatic instability (39). In the three lines carrying >300 CTG repeats, we observed very large differences between tissues in the size of CTG repeats (Fig. 5). These results show that there is also a size threshold for somatic instability between 20 and 55 CTG.
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In the three DM300 lines, mosaicism is barely detectable at birth but increases considerably with age (Fig. 5). As in DM patients and in the DM55 mice, the somatic instability in tissues tends towards expansion. In tissues with extensive mosaicism, somatic instability was first detected as a continuous smear extending towards larger CTG repeats. Then, with aging, most of the cells present larger CTG repeats detected on gels as bands, with increases that may reach >100 CTG (see, for example, the liver and pancreas in line 1177, Fig. 5A). Thus, somatic instability continues throughout life, with small progressive increases. Interestingly, in the livers of line 1177 mice, mosaicism is bimodal with a band corresponding to the ‘basal CTG repeat size’ (the size inherited from the parent observed in tissues in which instability was absent or undetectable such as blood, thymus, eyes and cerebellum) and a broad band corresponding to larger CTG repeats (Fig. 5A). These bands probably derive from different populations of cells in which the instability mechanisms are more or less active.
All the different mice analyzed in the same line presented the same pattern of mosaicism showing that, in any given line, there is no qualitative difference between individuals. However, we observed differences in the pattern of mosaicism between the DM300 lines. Surprisingly, the liver and kidney showed very different levels of mosaicism in the different DM300 lines. The liver, which presented the highest instability in DM55 lines, also showed considerable instability in the DM1177 line but not in the DM328 and DM1112 lines. For the kidney, instability was observed in the DM55, DM1177 and 1112 lines but was not detectable in the DM328 line. Southern blotting experiments confirmed the absence of very large amplifications that could have been missed by PCR in liver and kidney in the DM328 line (data not shown). In the DM1112 line, which carries three copies of the human fragment, the pattern of mosaicism was the same for all three repeats, whatever their size (see for example, in Fig. 5C, the two larger repeats of 390 and 190 CTG). Therefore, differences in somatic mosaicism between the three lines cannot be explained by differences in the size of the repeat.
Instability in the germline
In all DM300 lines we observed large CTG repeat expansions in the ovary and testis (Fig. 5). At 1 month of age in line DM300-328, expansions were not detectable in testis and barely detectable in ovary (data not shown). However, expansions were clearly observed in testis and ovary from mice older than 5 months of age (Fig. 5B). DNA extracted from purified spermatozoa showed the same mosaicism observed in testis. This mosaicism was much stronger in sperm than in blood, in which instability was undetectable (Fig. 5B). These results show that instability in the germline is also biased towards expansions in the gonads and (at least for males) in germ cells. This is consistent with the tendency towards expansion observed for intergenerational instability and with the increase in amplification with increasing male age.
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DISCUSSION |
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Comparison of intergenerational instability in transgenic mice and DM patients
In order to reproduce and to decipher the CTG repeat instability mechanisms involved in DM, we generated transgenic mice carrying large genomic DNA fragments cloned from DM patients. These fragments contained a normal repeat of 20 CTG, a 55 CTG amplification or an amplification of at least 300 CTG, surrounded by the original human DM flanking sequences. We studied the various lines produced over at least five generations and found that, despite the absence of very large amplifications in mice, the characteristics of intergenerational instability in DM patients and transgenic mice are very similar: (i) there is a threshold for intergenerational instability between 20 and 55 CTG in both DM patients and transgenic mice, which has been estimated to be ~40 in DM patients; (ii) the mutability of the repeat increases with its size. Even more strikingly, in lines carrying a full mutation, with >300 CTG, the percentages of amplifications (between 86.5 and 95% depending on the line) and contractions (between 2 and 5%) are very similar to those observed in DM families (11,12); (iii) the size variations are biased towards amplifications in the mice carrying the premutation of 55 CTG as well as in the mice carrying the full mutation of >300 CTG; (iv) amplifications depend on the sex of the transmitting parent and increase with the age of the male for paternal transmission. In DM, whether age of father at the birth of affected children has some consequence on the CTG repeat amplification has not been reported. However, an effect of the father’s age on CAG repeat instability has been reported in HD and DRPLA (45,49).
The influence of the sex of the transmitting parent on CTG repeat amplification in the offspring is particular in DM, which is the CAG/CTG repeat-associated disease with the highest level of instability. In diseases associated with CAG expansions and characterized by progressive neuronal loss, the CAG repeat is moderately amplified, the pathogenic length depends on the disease and does not exceed 200 CAG. For these diseases, there is a tendency towards larger amplifications after paternal transmission, with the maternal transmission giving smaller increases or contractions. This is also the case in DM for small repeats (<100 CTG), which give larger amplifications in the next generation following paternal transmission (11). This sex-specific difference could be explained by basic differences such as the number of cell divisions during male and female gametogenesis or by sex-specific differences in DNA metabolism and repair. However, for repeats of more than ~500 CTG, amplifications are larger after maternal transmission. Selection against larger alleles in sperm may account for this reversal of the sex-specific difference. The mechanism(s) involved in these sex-specific biases can now be investigated in our DM300 mouse model.
Factors affecting intergenerational instability
The DM300 model showed a frequency of contractions and amplifications similar to that observed in DM families. Furthermore, we consistently observed a bias towards expansion with both male and female transmission. These results contrast with those obtained with other mice models in which the frequency of repeat length changes was higher after female transmission, with, generally, a tendency towards contraction. Comparison of all the trinucleotide instability mouse models, including ours, allows distinction of properties for the CAG/CTG repeat instability: (i) the mutability, corresponding to the frequency of variations in the number of triplet repeats (expansions and contractions); (ii) the direction of mutability, towards expansion or contraction after male or female transmissions; and (iii) the range of size of amplifications.
In all mouse models generated so far, the mutability of the CAG/CTG repeat appears to be strongly correlated with the size of the repeat but also with the presence of human flanking sequences. Long repeats (>100 CAG/CTG) are very unstable in mice (40,41,46); however, human flanking sequences seem to be necessary to reproduce instability for moderate amplifications such as 55 CTG in our mice, 45 CAG in the YAC carrying the SBMA gene or 78 CAG in the cosmid carrying the DRPLA gene (39,44,45). It has been observed that, for the CAG repeat involved in Huntington’s disease (HD), the 48 repeats carried by a 4.6 kb fragment of human genomic flanking DNA are moderately unstable in transgenic mice, with 2% of meioses resulting in repeat changes. Interestingly, this 48 CAG repeat shows a similar frequency of mutation in knock-in experiments and a larger repeat of 109 CAG has a higher mutation frequency (73%) (46). These results also demonstrate the determinant effect of the size of the repeat for trinucleotide repeat mutability. In addition, comparison of these knock-in models with transgenic mice carrying stable 79 CAG repeats (37) suggests that, to some extent, the mouse hd cis-sequences allow some mutability of the CAG repeat. Such mutability probably depends on cross-species conservation of sequences and/or functional elements (like origin of replication) involved in the instability mechanisms. This cross-species conservation may differ for the various loci involved in trinucleotide diseases.
The direction of mutability may depend mainly on the human flanking sequences. This is illustrated particularly by comparison of our models and the CTG repeat transgenic mice developed by Monckton et al. (40). These mice, carrying only ~1 kb of human sequences but a large repeat of 162 CTG, have a high rate of somatic and intergenerational mutability (up to 70%, depending on the line and the integration site) (40,50). However, as female transmission generally results in contraction in these mice, the direction of change is different from that observed in human and in our mice carrying larger human genomic DNA. The direction of mutability depends also on other factors (sex-specific selection of large repeats or sex-specific DNA processing), the effects of which are probably mediated by cis-acting sequences. The precise effect of cis-sequences on triplet repeats is unknown. Brock et al. (25) demonstrated that the most unstable expanded CAG/CTG repeats are located within CpG islands and that the relative expandability of the repeats was strongly correlated with the GC content of the flanking sequences. The GC-rich chromatin environment could affect in vivo the flexibility of the repeat and the stability of abnormal structures or the machinery involved in DNA metabolism (repair, replication, etc.). Furthermore, in mammalian cells, DNA replication often initiates at CpG islands (51). The proximity of origin of replications near the trinucleotide repeats involved in diseases may affect their stability as has been demonstrated in bacteria and yeast models (27,28).
Even with a large amount of human genomic DNA surrounding the repeat, the third characteristic (range of amplifications) remains moderate in our models, in the mice carrying 45 CAG in the AR YAC (44) and in the transgenic mice carrying 78 CAG in the DRPLA gene (45). In all CAG repeat models, the range of amplification is smaller in mice and there is often a tendency towards contraction after female transmissions. Using a large repeat surrounded by extensive human genomic flanking sequences, we obtained a higher range of expansions, and CTG repeat instability was remarkably similar in its characteristics and dynamics to the CTG repeat instability observed in DM patients. However, even with > 300 CTG, the largest amplification observed in a single generation was 60 CTG. Enlargements of several hundred repeats (or ‘big jumps’), which are observed in DM families, were not observed in mice. If intergenerational instability results from the mosaicism observed in the germline, with enlargement of the CTG repeat throughout life, then the lower level of amplification in mice may result from their shorter reproductive life-span, as suggested previously (45). Alternatively, the mechanisms involved in trinucleotide repeat instability may act at a greater repeat length in mice than in humans. The DNA repair system may be more efficient and the repeat size threshold for ‘big jumps’ may be higher in mice. We found a negative correlation between the size of the repeat and the range of expansions after male but not after female transmission. Therefore, we will continue to breed DM300 transgenic females to determine the extent to which the repeat can be expanded in mouse and whether a threshold can be reached to obtain big jumps in amplification.
Somatic instability in transgenic mice
Like DM patients, the DM55 and DM300 mice exhibit size- and age-dependent somatic instability with a bias towards expansion. Instability mechanisms linked to replication and transcription have been proposed from studies in bacteria, yeast and mice models. As we reported previously, differences in the replication rates and transcription of the genes included in the region close to the repeat are not sufficient to explain the differences in the level of somatic mosaicism between tissues (47). Replication-independent mechanisms have been proposed for CAG instability in oocytes (42). These mechanisms may also, together with replication-based mechanisms, be involved in somatic trinucleotide repeat instability. The open chromatin associated with transcription, rather than transcription itself, seems more likely to be required for instability and could facilitate unusual structure of the repeat or facilitate access to the machinery involved in somatic instability. Cis-sequences and integration sites may also affect the somatic mutability of the repeat. Although we observed no differences in the somatic mosaicism patterns of the DM55 lines, we found that the level of instability differed between DM300 lines, particularly in the liver and kidney. The size of the repeat, which affects intra-tissue instability, cannot account for the differences between tissues in the different lines. The DM300-328 and DM300-1177 lines lack ~1 and ~16 kb of the 5' end of the integrated fragment, respectively (Fig. 1). This suggests that the loss of these sequences and/or the different integration sites may affect somatic instability in the liver and kidney although, surprisingly, this does not seem to affect somatic instability in other tissues.
Concluding remarks
It has been suggested that during replication the formation of a hairpin on newly synthesized Okazaki fragment favors expansion. The formation of large hairpin structures in repeats larger than Okazaki fragments may favor the large saltatory expansions (31). Alternatively, long repeats may increase the formation of double-strand breaks after the stalling of the replication forks, resulting in expansion after repair (52,53). Several candidate proteins may be involved in instability mechanisms. FEN1, a nuclease with the ability to remove the 5' flap of the Okazaki fragment, may not be able to process trinucleotide hairpins, resulting in expansion (30,53,54). Other proteins involved in various DNA repair systems, such as DNA mismatch repair or double-strand DNA break repair, may also be involved in generating the trinucleotide amplifications observed in diseases. Recently, Manley et al. (55) demonstrated that Msh2 is involved in the CAG somatic instability in transgenic mice for expanded HD exon 1. Mouse models like ours, showing a high level of instability tending towards expansion, as in patients, also provide a powerful tool for testing the involvement of these candidate genes in intergenerational and somatic instability. Crossing our mice with DNA repair gene knockout mice (when available) should provide further insight into the mechanisms involved and should help to determine which DNA repair systems are involved in intergenerational and/or somatic instability.
There is now circumstantial evidence that CTG expansions may affect the expression of neighboring genes and that abnormal DMPK RNA may have a detrimental effect on RNA metabolism (56–58). Careful phenotypic analyses are underway to determine which symptoms result from the presence of abnormal DMPK messengers. It is not clear whether the somatic instability increasing with age in DM is correlated with the progression of the disease in patients. However, DM300 mice may be useful for testing triplet repeat-based therapy aimed at preventing somatic expansion or reducing the deleterious effects of abnormal DMPK RNA.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONResultsDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cosmid libraries
DNA extracted from lymphoblastoid cell lines were partially digested with MboI and fractionated by preparative pulse-field gel electrophoresis. Fragments (40–45 kb) were electroeluted from the gel and ligated in the SCossbs vector linearized with BamHI. Scossbs derives from SCos1 vector modified by insertion of a polylinker containing SalI, BamHI, SalI restriction sites. Ligation products were packaged with the Stratagene (La Jolla, CA) Gigapack II gold packaging extract and bacteria were plated on 20 x 20 cm selective plates. Two replica filters were prepared for each plate and hybridized with DMHR4/5 probe. This probe was synthesized by PCR using genomic DNA and DMHR4/DMHR5 primers as described previously (39). NdeI–ClaI restriction fragments from clones DM20 and DM300 were purified on 0.8% agarose gel and ligated in DM55 clone digested by NdeI–ClaI. Multiple digests with restriction enzymes were used to check integrity of the clones.
Production of transgenic mice
DM20, DM55 and DM300 cosmids were digested with SalI. The 45 kb genomic inserts were electroeluted from agarose gel and finally purified as described previously (39). Transgenic mice were produced by microinjection of the purified insert (0.4 ng/µl) into pronuclei of fertilized mouse eggs (B6D2/F1) that were subsequently transferred into foster mothers. Transgenic mice were screened by PCR using DNA prepared from tail biopsies and primers DMHR4/DMHR5. Positive mice were confirmed by Southern blotting after digestion with BamHI and hybridization with the BamHI 1.4 kb fragment containing the DMPK CTG repeat. Transgenic animals were crossed with C57BL/6 mice.
Preparation of sperm DNA
Sperm DNA was extracted according to Jeffreys et al. (59). Briefly, semen was recovered from the vas deferens, diluted with 100 µl of 1x SSC and incubated for 30 min at 37°C. After centrifugation, the pellet was resuspended in 100 µl of 1x SSC, 1% SDS and incubated for 15 min at room temperature to lyse seminal leukocytes and epithelial cells. Sperm heads were pelleted by centrifugation for 4 min at 12 000 r.p.m., rinsed twice with 150 µl of 1x SSC, 1% SDS, rinsed once with 150 µl of 1x SSC and resuspended in 100 µl of 0.2x SSC. Sperm heads were lysed by the addition of 2-mercaptoethanol to 1 M and SDS to 1%, and after incubation for 1 h at room temperature, DNA was phenol extracted.
DNA analysis
The transgene copy number was determined in each line by Southern blot hybridization after BamHI digestion. Southern blots were realized with transgenic tail DNA and control mouse DNA mixed with known DM55 fragment copy number. P59 (2 kb PstI fragment of the DMPK gene; Fig. 1) and EPO (1 kb PstI fragment from the mouse erythropoietin gene) were used as probes. The copy number in transgenic mice was deduced by densitometric comparison of the ratio between the DMPK and EPO fragments with the known standards. Quantification was performed by PhosphorImager scans (Molecular Dynamics) using the IMAGEQUANT version 1.1 image analysis program. The same blots were hybridized with extC 7.1 to map the junction fragments and with the DM55 cosmid (digested with BamHI) to check for the transgene integrity.
CTG repeat analysis
To determine CTG repeat lengths in the DM20 and DM55 transgenic mice, PCR analyses were performed in 50 µl reactions containing 16.6 mM (NH4)2SO4, 67 mM Tris–HCl pH 8.8, 1.5 mM MgCl2, 67 µM EDTA, 10% dimethylsulfoxide (DMSO), 10 mM 2-mercaptoethanol, 0.2 mM dNTPs, 0.2 µM primers 101 and 102 (101, 5'-CTTCCCAGGCCTGCAGTTTGCCCATC-3'; 102, 5'-GAACGGGGCTCGAAGGGTCTTGTAGC-3'). Cycling parameters were 94°C for 6 min, 65°C for 1 min, 72°C for 1 min, followed by 30 cycles of 94°C for 1 min, 65°C for 1 min, 72°C for 1 min. Amplified product (2 µl) was mixed with 2 µl of formamide loading buffer, heated for 5 min at 100°C, separated on 6% denaturing acrylamide gel, blotted on nylon membrane and hybridized with 3'-end-labeled 101 primer. To determine the CTG repeat length in DM300 transgenic mice, 15 ng of tail DNA samples were amplified in 25 µl reactions using a previously described buffer system (60), 1.4 µM primer 101, 1.4 µM 102 and 0.2 U of Taq polymerase (Gibco BRL, Gaithersburg, MD). The DNA was denatured by heating to 95°C for 10 min. Reactions involved 30 cycles of 94°C (45 s), 66°C (45 s) and 70°C (3 min) with a chase of 66°C (1 min) and 70°C (10 min) in a Perkin Elmer 9600 thermal cycler. Amplified product (3 µl) was mixed with 2 µl of formamide loading buffer, heated for 5 min at 100°C and subjected to electrophoresis in a 4% denaturing acrylamide gel at 25 W for 16 h. After electrophoresis, the gel was incubated in 1x TBE with ethidium bromide for 10 min and UV exposed for 8 min. DNA was blotted onto nylon membrane and probed with the 3'-end-labeled 101 primer. Hybridized bands were detected by autoradiography and their size determined by comparison with M13 sequencing reactions (DM20, DM55) or with a 100 bp DNA ladder (DM300).
Statistical analysis
Statistical analyses were performed using the StatView program (SAS Institute Inc.).
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Acknowledgements |
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This work was supported by grants from INSERM, the Association Française contre les Myopathies (AFM), the Délégation à la Recherche Clinique (DRC), the Assistance Publique-Hôpitaux de Paris, the Leg Poix and the Université René Descartes. H.S. and A.-S.L.-B. were supported by grants from AFM and G.G. by a grant from the DRC.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +33 1 44 49 45 23; Fax: +33 1 47 83 32 06; Email: gourdon@necker.fr
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REFERENCES |
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