Human Molecular Genetics, 2001, Vol. 10, No. 8 845-854
© 2001 Oxford University Press
Mouse tissue culture models of unstable triplet repeats: in vitro selection for larger alleles, mutational expansion bias and tissue specificity, but no association with cell division rates
Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, Anderson College, 56 Dumbarton Road, Glasgow G11 6NU, UK
Received 18 December 2000; Revised and Accepted 19 February 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The expansion of CAG·CTG trinucleotide repeats has been associated with an increasing number of human diseases. Once into the expanded disease-associated range, the repeats become dramatically unstable in the germline and also throughout the soma. Instability is expansion-biased, contributing towards the unusual genetics, and most likely the tissue-specificity and progressive nature of the symptoms. Such expansions constitute a unique form of dynamic mutation whose mechanism is poorly understood. It is generally assumed that repeat length changes arise via replication slippage, yet no direct evidence exists to support this hypothesis in a mammalian system. We have previously generated transgenic mouse models of unstable CAG·CTG repeats that reconstitute the dynamic nature of somatic mosaicism observed in humans. We have now used tissues from these mice to establish in vitro cell cultures. Monitoring of repeat stability in these cells has revealed the progressive accumulation of larger alleles as a result of repeat length changes in vitro, as confirmed by single cell cloning. We also observed the selection of cells carrying longer repeats during the first few passages of the cultures and frequent additional selective sweeps at later stages. The highest levels of instability were observed in cultured kidney cells, whereas the transgene remained relatively stable in eye cells and very stable in lung cells, paralleling the previous in vivo observations. No correlation between repeat instability and the cell proliferation rate was found, rejecting a simple association between length change mutations and cell division, and confirming a role for additional cell-type specific factors.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The expansion of unstable trinucleotide sequences in the human genome is the primary genetic defect associated with a particular class of complex human diseases, including myotonic dystrophy type 1 (DM1), Huntington’s disease (HD), fragile X syndrome, Friedreich’s ataxia (FRDA) and an ever increasing number of spinocerebellar ataxias (SCAs) (1). Most of these disorders, such as DM1, HD and the SCAs, are associated with the expansion of CAG·CTG repeats. In addition to a common genetic defect, these conditions also share an unusual pattern of inheritance known as genetic anticipation, that is, a decreasing age of onset and worsening of symptoms through successive generations. Moreover, the repeats are also somatically unstable and inter- and intra-tissue differences in repeat length distributions are commonly observed.
Of all of the CAG·CTG expansion disorders, DM1 presents the widest range of expansion sizes between patients and is the disorder for which somatic mosaicism is most fully characterized. DM1 is an autosomal, dominantly inherited disorder characterized by muscle weakness, wasting and myotonia, but also including a variety of additional symptoms with a wide range of ages of onset (2). DM1 is associated with the expansion of a CTG sequence within the 3'-untranslated region (3’-UTR) of the DM protein kinase (DMPK) gene on chromosome 19 (3–8). The expanded DM1 alleles exhibit extensive germline and somatic instability, biased towards expansion (5,9–16). Interestingly, larger repeat lengths are consistently observed in the major affected tissue, muscle, relative to blood DNA (11–13,16). In addition, longer average repeat lengths and broader ranges of variability have been found in older patients (16–18). The size of the inherited expanded allele and the age of the patient have been identified as critical factors controlling the degree of somatic mosaicism (17,18). Given that expansion-biased germline instability accounts for the phenomenon of genetic anticipation (19), it seems likely that the characteristic expansion-biased, age-dependent, tissue-specific dynamics of the repeat in the soma accounts, at least in part, for the progressive nature and tissue specificity of the symptoms in DM1. Moreover, Kennedy and Shelbourne (20) have recently revealed striking large expansions restricted to the affected tissue, striatum, in a knock-in mouse model which suggests that somatic mosaicism similarly contributes to pathogenesis in HD.
It is generally assumed that variability arises through DNA replication slippage during cell division (21,22). Such a mechanism might be facilitated by the propensity of these sequences to adopt unorthodox non-B-DNA structures, such as slipped-strand DNA (S-DNA) (23), which could induce polymerase stalling and/or stabilize replication intermediates. Attractive as this suggestion is, there is, as yet, no direct evidence from a mammalian system to support the existence of such structures in vivo, nor the hypothesis that mutations arise during DNA replication. Indeed, there are no obvious relationships between tissues in which the repeats are prone to change and rates of cell turnover (11–13,16,20,24–26). However, it should be considered that such analyses have been primarily performed on whole tissues or organs comprised of multiple cell types likely to have very differing dynamics in terms of both cell turnover and repeat metabolism. Such a complex scenario confounds attempts to make simple correlations and may have obscured some more subtle relationships.
In order to gain greater insight into the mechanisms underlying triplet repeat dynamics, trinucleotide tracts have been cloned into simple model organisms such as Escherichia coli (27) and Saccharomyces cerevisiae (28). These systems have revealed some important insights into the factors affecting repeat dynamics, such as orientation (putatively with respect to replication origins), transcription and DNA repair gene mutations. However, the repeats are inherently biased toward contraction in such systems, in complete contrast to the predominantly expansion-biased behaviour observed at most loci in humans. So far the analysis of repeat stability in cell cultures of patient-derived cells has yielded mixed results. The DM1 repeat continued to expand in vitro in primary cells derived from a DM1 fetus (29). In contrast, Epstein–Barr virus (EBV)-transformed lymphoblastoid cell lines (LBCLs) derived from DM1 patients adopt an unusual pattern of repeat variability (30). In addition to the frequent small length change mutations that are biased toward expansion (31), rarer, but very large, deletion mutants are also observed at a frequency exceeding that detected in vivo. These results suggest that the EBV-transformation process results in altered cellular DNA metabolism. Such an effect might be expected given that some EBV proteins alter the transcriptional activity of cellular genes involved in DNA processing (32). Recently, it has been reported that expanded GAA repeats at the FRDA locus in EBV-transformed LBCLs display contractions and expansions of similar magnitudes and frequencies (33). Thus, it would appear that although EBV-transformed LBCLs may be useful for modelling some aspects of repeat metabolism, their failure to faithfully recreate in vivo dynamics limits their overall utility. Moreover, although primary human cell cultures may be a good model, their availability from individuals with rare inherited disorders is severely limited.
In order to create additional model systems in which repeat biology may be assessed in vivo in a mammalian system, we (34) and others have created transgenic mice containing unstable expanded CTG·CAG arrays (26,35–39). To examine the intrinsic stability of CAG·CTG repeats, we generated five transgenic mouse lines carrying a portion of the DMPK 3'-UTR with 162 CTG repeats, derived from the human DM1 locus. One line in particular, Dmt-D, which comprises a single copy of the construct, has been shown to reproduce the dramatic tissue-specific, age-dependent and expansion-biased repeat instability associated with somatic mosaicism in DM1 patients (25). Similar mutational dynamics have been reported in transgenic models carrying large 45 kb fragments of the human DM1 locus, incorporating approximately 300 repeats (26). Transgenic mouse models that mimic the trinucleotide somatic instability observed in patients represent a good tool to resolve the molecular mechanisms regulating repeat dynamics in a mammalian environment. The development of a cell culture system from such animals which faithfully reproduces the in vivo trinucleotide dynamics in vitro will create new avenues to investigate the multiple factors affecting the dynamics of repetitive sequences under controlled conditions. To this end we have sought to establish cell cultures from tissue samples harvested from Dmt-D transgenic mice and monitor the CTG repeat over extensive time periods, with the aim of clarifying the effect of a large number of cell divisions on trinucleotide repeat stability. Such a model system should allow us to provide new insights into the cellular metabolism of the simple CAG·CTG tandem repeats associated with inherited human disease.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Growth dynamics of mouse cell cultures
In an attempt to establish primary mammalian cell cultures in which the dynamics of expanded CAG·CTG repeat tracts could be monitored and investigated in vitro, we harvested lung, eye and kidney from a 6-month-old male Dmt-D transgenic mouse. Using either the explant technique (eye) or enzymatic dissociation procedure (lung and kidney), mouse cell cultures were successfully established. Following an initial period characterized by low growth rates, all the cell cultures entered a continuous exponential growth phase, proliferating at similar and constant rates (Fig. 1). The cell proliferative capacity of each culture was estimated according to the observed population doubling time (PDT), which was calculated based on the cell counts determined at each passage (Table 1). The late rapid cell growth observed in vitro is consistent with the spontaneous immortalization of cells, which is known to occur at a relatively high frequency with mouse cell cultures (40,41). The time taken to reach peak growth rate was longest for the kidney culture, which took
120 days, in contrast to the lung and eye cell cultures, which took 60 days.
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Characterization of cultured cell types
Each culture consisted of an initial heterogeneous cell population, comprising cells with clearly different morphologies and probable distinct proliferative capacities. The cultures soon became more homogeneous, exhibiting prevalent spindle morphology typical of fibroblasts after less than five passages (Fig. 2). The nature of the cultured cells was confirmed immunocytochemically by staining with primary antibodies directed against vimentin and cytokeratins (42). All the cultures were positive for vimentin and negative for cytokeratins, consistent with a fibroblastic rather than epithelial phenotype (Fig. 2). Nonetheless, the precise morphology was clearly distinct between the cultures derived from the different tissues, indicating a different absolute origin for each culture.
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Tissue-specific trinucleotide instability and selection for longer alleles in cultured mouse cells
Following the establishment of these tissue-specific Dmt-D murine cell lines, DNA samples were collected at every passage and transgene repeat length variability was assessed by small pool PCR (SP-PCR) analysis (16,43). The length of the repeat in the cultures and the level of variation were compared with the progenitor allele length in the donor mouse (173 repeats, as determined by PCR length analysis of tail DNA at weaning) and the level of variability present in the tissue from which the culture was originally derived.
Variability in lung cell cultures.
The lung tissue from which the culture was established showed relatively low levels of variability, with most alleles (>90%) remaining within ±10 repeats of the progenitor allele (173 repeats) (Fig. 3A). After 15 doublings, the lung cell culture displayed an even lower level of variability, with the vast majority of cells within ±5 repeats of the predominant allele (about 175 repeats). The reduction in variability observed in the progression from in vivo to in vitro suggests that only a very few cells grew in culture. Indeed, the degree of repeat length homogeneity observed in the culture and the relative increase in size detected suggest that this culture may have been taken over very quickly by derivatives of a few, or possibly even only one, of the cells present in the original tissue carrying a slightly larger allele. Surprisingly, even after as many as 100 or 200 doublings in vitro (>300 days), the level of repeat variability remained very low, with only a small increase in average allele length up to 177 repeats. The maintenance of such a low level of variability in vitro after so many doublings indicates that mammalian cells are capable of faithfully replicating large expanded CAG·CTG repeat tracts through many cell divisions, even at a locus that is extremely unstable in other cells. Also of note was the relatively late appearance in the culture of a subset of cells carrying a deletion of approximately 30 repeats relative to the major allele in the culture. After 200 doublings, these cells comprised 10% of the cell population, but rose to 25% by 250 doublings. Presumably, this shift was mediated by drift and/or selection within the cell population rather than repeated mutations to the same length allele. This assertion is supported by the bi-directional nature and different sizes of similar shifts observed in other cultures (see below).
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Variability in eye cell cultures.
Higher levels of instability were detected in cultured eye cells (Fig. 3B). Once again, very early in the establishment of the culture, the range of variability observed was very different to that detected in the progenitor tissue. By as few as five doublings, the predominant cell population contained an average allele length of around 185 repeats, 12 repeats larger than the major allele in the original tissue (173 repeats). By 10 doublings, a distinct subpopulation of cells appeared with an average size of approximately 210 repeats. This population presumably represents clonal expansion of a rare cell carrying a large expanded allele, either present in the original tissue or having arisen as a spontaneous mutant in vitro during one of the earlier passages. Nonetheless, by 10 doublings each population showed moderate size variability, characterized by small changes mostly limited to fewer than ±5 repeats around the average repeat length. The moderate instability was maintained, with the level of variability and the average allele length gradually increasing within each of the two main populations of cells. This effect was very clear up to 70 doublings, by which time the average allele size in each population had risen by a further five repeats relative to that observed at 10 doublings. After 110 doublings, however, the proportion of cells in the population carrying the larger alleles started to decrease. By 240 doublings in vitro, the eye cell population initially carrying about 185 repeats had increased in average allele length to approximately 195 repeats, but had overgrown the culture, causing a reduction in overall repeat length variability.
Variability in kidney cell cultures.
Kidney is the tissue that shows the highest levels of variability and the largest expansions in vivo. Repeat variability within the original tissue showed the typical trimodal distribution highly biased towards expansion (25) (Fig. 3C). Most cells carried repeats within the first peak of variability with alleles within –5 to +10 repeats of the progenitor (173 repeats). Additional peaks of variability were observed most clearly at approximately 200 and 230 repeats. Although a relatively high level of variability was retained after five doublings in vitro, the predominant alleles from the first peak of variability detected in the original kidney tissue were entirely absent. It appears as if only cells from the second and third peaks were able to grow in vitro. Indeed, by as few as 20 doublings, the second peak of cells had disappeared and only cells carrying the largest repeats were maintained. Single molecule analyses allowed us to quantitatively define more clearly the progression of repeat variability in cultured kidney cells (Fig. 4). After 20 doublings a narrow range of repeat sizes was defined, with an average repeat length of about 260 repeats, which not only corresponded to the largest repeats initially found in vivo, but also overlapped with the largest peak described above after five doublings in culture. After 50 doublings, a pattern of instability very reminiscent of the highly positively skewed distributions observed in vivo in both humans (16) and mice (25) was observed, with very few alleles being detected below the lower boundary of 250 repeats. The level of repeat variability peaked at this stage, with a mean allele length of 270 repeats and with 5% of cells containing alleles greater than 300 repeats in length. However, the repeat length heterogeneity was reduced dramatically and the mean allele length decreased to 260 repeats after 100 divisions in culture. A similarly low level of variability was retained even after 150 population doublings. The repeat dynamics in cultured kidney cells suggests the development of an expansion-biased mechanism in vitro, which may be disturbed by population fluctuations, such as the dramatic selective sweeps described at 20 and 100 doublings.
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It is important to note that no obvious relationship was found between the repeat instability in the three tissue specific cell lines analysed and their proliferative capacity, as assessed by the PDTs (Table 1). Taken together, the results reported above for the three different tissues, support the maintenance of in vivo tissue-specific trinucleotide instability in vitro, which is expansion-biased, particularly during the early stages of the eye and kidney cell cultures.
Accumulation of mutations in single cell-derived clones from kidney cell cultures
To discount the possibility that the variability detected in culture could be an artefact derived purely from an in vitro selection process for the cells harbouring longer alleles, rather than the outcome of the intrinsic instability of the transgene in vitro, clonal lines derived from single cells were established. The clones were isolated by limiting dilution from the kidney cell culture after 20 population doublings, grown for a further 20 doublings, and the CTG repeat variability determined by SP-PCR (Fig. 5). All the clones exhibited repeat size heterogeneity, which corroborates the progression of somatic instability in culture. In addition, they showed different average repeat sizes and different ranges of repeat distribution. For one clone in particular (clone 2), a very broad range of repeat sizes was observed. Such a difference could not be accounted for by cell division rates, since no significant variations in the proliferative capacities were observed between the three clones studied here (data not shown).
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Age-of-donor effect on trinucleotide instability observed in culture
To investigate the influence of the age of the donor mouse at death on the trinucleotide instability detected in culture, kidney cell lines were established from two younger Dmt-D male mice, aged 5 weeks and 3 months, and one very old mouse aged 30 months. The two cultures from the young mice showed similar growth rates, with PDTs of 42 and 45 h, respectively (Table 1). Consistent with the age-dependent accumulation of somatic mosaicism in vivo, the youngest mouse analysed in this study displayed very little somatic variation in the original tissue from which the cell line was derived, with most cells with an allele size of approximately 160–175 CTG repeats (Fig. 6A). As with previous cultures though, the level of variability was almost immediately reduced in the first few passages and by five doublings the average allele size was about 165 repeats with most variants within ±5 repeats. The range of variation and average allele length increased up to 35 doublings, by which point two major populations of cells with approximately 170 and 185 repeats were present. After an additional 30 doublings the cells containing the larger alleles predominated, accompanied by an overall reduction in the range of variability, reflective of a selective sweep. The repeat sizes ranged from about 185 to 215 repeats in the original kidney tissue of the 3-month-old mouse, intermediate between that observed in the 5-week- and 6-month-old mice (Fig. 6B). Once again, variability was rapidly reduced in culture; most notably, by 20 doublings the average allele length had increased to approximately 195 repeats, but with a reduced overall range. By 35 doublings a second major population of cells carrying approximately 225 repeats appeared. This population took over the culture completely by 65 doublings and increased in length up to around 240 repeats. The level of variation present in vivo in the very old mouse was, as expected, very high with a small subset of cells carrying alleles as large as 720 repeats (Fig. 6C). Nonetheless, the most predominant cells contained expansions in the range of 160–180 repeats. However this high level of variability was massively reduced very early in the establishment of the culture. By five passages only three major, but highly distinct, populations persisted, suggesting that this culture was most likely derived from only three progenitor cells. Once again the allele sizes present in the cells that predominated in culture were much larger than those present in the majority of cells in vivo. Overall, these data confirm the proliferative advantage in vitro of cells that contained large repeats in vivo. They also indicate that the expansion-biased progression of somatic instability in vitro is a reproducible phenomenon using kidneys from Dmt-D mice as a source material and that selective sweeps are a common occurrence. These data also appear to support an effect of the age of the mouse at death on the stability of the transgene in culture, with the repeat appearing to be less stable in cell lines derived from older mice than those from younger mice. However, this may be a result of the longer allele lengths that predominate in the cultures derived from older mice, rather than a true age-of-donor effect on repeat stability.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The expansion of simple repetitive trinucleotide sequences is causally involved in the molecular bases of an increasing number of human diseases. In the soma, repeat instability is expansion-biased, tissue-specific and age-dependent; dynamics that are consistent with a role in the tissue specificity and progressive nature of the symptoms. However, little is known about the mutation processes that give rise to somatic mosaicism. To facilitate the study of somatic repeat instability in a mammalian system, we have previously generated transgenic mouse models of unstable CAG·CTG trinucleotides (34), one line of which (Dmt-D) replicates gross age-dependent, tissue-specific, expansion-biased somatic mosaicism (25). We have now harvested tissues from these mice to establish cell lines and monitored repeat stability over a long period under conditions of rapid cell proliferation. These investigations have revealed that the dynamic pathway observed in vivo, i.e. accumulation of multiple small mutations biased toward expansions, is conserved in an in vitro mammalian cell model. Most interestingly, the tissue specificity observed in vitro also reflects that observed in vivo. In particular the repeat remains remarkably stable in lung cells, even after several hundred days and hundreds of cell divisions in culture. In contrast, the repeat is very unstable in cultured kidney cells. These tissue-specific differences are observed, despite the fact that the PDT for cell lines from the three different tissues is similar. It has been postulated that mitotic cycles generate somatic heterogeneity of repeat lengths. According to this model, we would predict that the higher the cellular proliferation rates, the higher the variability in repeat number generated. Although the tissue specificity of somatic mosaicism in vivo shows no obvious correlation with the known rates of cell turnover, it could be argued that the multi-cell-type complexity of whole tissues might have masked such a relationship. The results from our in vitro model demonstrate conclusively that variation in repeat stability cannot be accounted for simply in terms of PDTs and must be related to other cell-type specific effects. Cell lines homogeneous for cell-type, such as those described here, should greatly facilitate the identification of such factors.
Surprisingly, cells carrying longer repeats were selected during the first few passages of all of the cultures, quickly giving rise to a larger average size than observed in the progenitor tissue. Accompanying this increase was an overall reduction in variability, suggesting that each cell line was founded from a very small number of cells. The probable explanation for this effect is the selective growth advantage in culture of cells that already contained a longer repeat in vivo. Alternatively, it is possible that the highly proliferative cells from which the cultures were established had a very high rate of expansion during their establishment. However, the fact that this effect was observed in all of the cultures, but was most prominent in cultures established from tissues with a high degree of mosaicism already established in vivo, suggests that the former possibility is most likely. Similarly, EBV-transformed LBCLs harbour larger repeats than peripheral blood leukocytes (30), also supporting a growth advantage in vitro of cells that contained longer repeat tracts in vivo. The factors that might co-facilitate repeat expansion in vivo and rapid proliferation in vitro remain unknown.
Selective sweeps are not restricted to the early stage of the culture, and continue to occur at later passages. These later sweeps can similarly result in dramatic shifts in repeat length distributions. For instance, the shift from a mean allele size of approximately 195 repeats after 20 doublings in the kidney cell culture from the 3-month-old mouse to about 240 repeats after 65 doublings does not appear to result from the gradual accumulation of multiple mutations in a homogeneously evolving population (Fig. 6B). Rather, it appears that at some point in the culture a rare cell arose with a markedly larger allele than the average, and that derivatives of this cell had a selective advantage, eventually taking over the culture completely. Other less dramatic selective sweeps have been observed at late passage in these cell lines, but they are not always in favour of the cells carrying larger alleles. For instance, the bimodal distribution present in the eye cell culture after 70 doublings (peaks at 190 and 210 repeats), resolved to a unimodal population of about 200 repeats by 240 doublings (Fig. 3B). Presumably, therefore, the favourable advantages gained by the selected cells resulted from the acquisition of additional mutations elsewhere in the genome, rather than any direct effect of the repeats on cell proliferation. Similar selective sweeps have been observed in human EBV-transformed LBCLs carrying GAA expansions at the FRDA locus (33) and CTG expansions at the DM1 locus (31). Whilst the selective sweeps observed at the GAA expansion in the FRDA cells were not obviously biased in their direction, those at the DM1 locus were associated with a growth advantage of cells carrying longer repeats. Indeed, the authors have suggested that there is a direct cause and effect relationship between the CTG expansion length at the DM1 locus and the proliferative capacity of the cell, presumably mediated by abnormal function of one of the genes flanking the endogenous repeat. Such an effect would not be expected in our system since the transgene contains no coding sequences, has been randomly integrated into the mouse genome and is not associated with any obvious phenotype in hemi- or homozygous Dmt-D mice. Such selective sweeps complicate attempts to provide an accurate measure of the repeat dynamics in any given cell line since the level of variation present can be rapidly reduced by selection. Moreover, the mutations that are selected will be those affecting cell growth and turnover and these might also be expected to indirectly or directly effect DNA metabolism. As such, it is probable that the dynamics of repeat metabolism will change with time. Indeed, it appears as if the repeats are most unstable in the early passages of the culture and become more stable with time. Thus, it appears as if the early passage cells are more in vivo-like, initially retaining the cell-type specificity of instability, but progressing toward a similar level of high stability. This further emphasises the requirement for a readily accessible source of tissue as afforded by the transgenic mice. Patient-derived human samples from individuals with rare conditions are not readily available. Moreover, the common genetic background of inbred transgenic mouse lines and their controlled environment reduces variability due to other factors that are not easily corrected for using human samples.
In addition to complicating attempts to quantify repeat dynamics in cell lines, the selective sweeps observed may have other, more general implications. We have been able to detect these sweeps because very high levels of variation are a feature of the system we assay. There is no reason to assume that such sweeps are not also a feature of other tissue culture systems and it is important that their potential effects are also considered when attempting to interpret the results obtained. This is particularly critical for attempts to study other aspects of triplet repeat biology such as effects on gene expression. It is standard practice to measure repeat length in either the progenitor tissue or at one time point in the culture and assume this remains constant. Our results indicate that such simple assumptions cannot be made.
Recently, Manley et al. (44) have reported the establishment of a similar mouse tissue culture system using tissues from the R6/2 mice transgenic for exon 1 of the HD gene carrying 154–155 CAG repeats. Rather than using primary cell lines though, they have established continuously passaged SV40-transformed fibroblasts. This system also provides evidence for selection accompanied by reductions in the level of variation observed. Expansion biased repeat instability was also observed, although the length changes recorded were all relatively modest (<15 repeats), even after 600 doublings in vitro. The higher levels of repeat heterogeneity detected in our system may result from either a higher intrinsic instability of the Dmt-D transgene, the dynamics of the repeat in the source tissues used or an effect of immortalization. SV40 expression clearly alters progression through the cell cycle with evidence for direct effects on aspects of the DNA repair and metabolism machinery (45). Alternatively, it may simply result from the higher sensitivity of SP-PCR in detecting rarer alleles compared with the electrophoretic profiles generated using high template levels, fluorescent primers, automated fragment analysis and GeneScan software (20,25).
These data strongly suggest that DNA replication is not the sole explanation for repeat instability, implying a role for additional cis and/or trans acting tissue-specific factors in the control of the dynamics of repetitive sequences in the soma. Additional evidence for non-cell division dependent instability is afforded by the age-dependent decrease in stability of maternally transmitted expanded CAG repeats in SCA1 and DRPLA transgenic mice (37,46) and the patterns of somatic mosaicism observed in various transgenic mouse models (for example, 20,25,26,35). One of the primary functions of the mismatch repair machinery is to ensure the faithful replication of DNA during cell division. Therefore, if replication slippage were the primary mechanism by which mosaicism arose, it would be expected that loss of function of mismatch repair would enhance repeat instability. In direct contrast to this hypothesis, however, it has been reported recently that the mismatch repair gene Msh2 is actually required for the accumulation of somatic mosaicism in the expanded CAG HD exon 1 repeat in R6/2 transgenic mice (47). Consequently, a mutational process relying on the repair of DNA presents as an alternative hypothesis. The substrate for repair could be either damaged DNA, such as might be induced by oxidative damage, or the adoption by the repeats of alternative non-B-DNA structures such as S-DNA. Indeed, it has been demonstrated previously that S-DNA is recognized and bound by MSH2 (48). Both of these mechanisms could operate in non-dividing cells. Thus, tissue-specific instability could be mediated, not by replication differences, but by either post-replicative DNA repair efficiencies, the level at which DNA damage is acquired or the rate at which non-B-DNA structures are formed.
In summary, we have established cell lines derived from a transgenic mouse model for triplet repeat instability and demonstrated that they retain the tissue-specific, expansion-biased instability observed in vivo. Under conditions of rapid cell turnover, our data support an expansion mechanism that might not be strictly dependent on cell division, contrasting with the prevalent DNA polymerase slippage hypothesis. This readily renewable primary cell system creates novel and exciting avenues to study the complex dynamics of triplet repeats.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mouse tissue samples
Dmt-D transgenic mice were killed by cervical dislocation and the kidneys, eyes and lungs were removed and kept on ice for up to 1 h until processed. All mice were hemizygous for the transgene on a pure FVB/N background.
Establishment and maintenance of cell cultures
Depending on the tissue, the primary cultures were established either by the explant technique (eye cultures) or enzymatic dissociation (lung and kidney). The eyeballs were transferred to 100 mm dishes, rinsed and dissected in PBS. The tissue was placed cell-side down on a 60 mm dish and minced into small fragments (1 mm2). All pieces of tissue were flattened and dispersed on the dish. Culture medium was gently added and the culture was incubated at 37°C in a humidified 5% CO2 atmosphere. The standard growth medium consisted of Dulbecco’s modified Eagle’s medium (DMEM) with high glucose, supplied with 10% fetal bovine serum, 100 U ml–1 penicillin and 100 µg ml–1 streptomycin (Gibco BRL Life Technologies). Once cells became 60–80% confluent they were subsequently subcultured at a ratio of 1:5 or 1:10 during the first five passages and kept under the same conditions. The enzymatic dissociation of kidney and lung tissues was essentially performed as described previously (49). The organs were transferred to 100 mm dishes containing 10 ml sterile PBS and minced into 1 mm3 cubes. The minced tissues were transferred into 15 ml Falcon tubes and washed twice with sterile PBS. The tissues were digested with 5 ml of 0.25% trypsin solution in a 37°C humidified 5% CO2 incubator for 30 min with limited shaking. After the incubation the pieces of tissue were allowed to settle down and the supernatants were transferred into fresh 15 ml tubes. The cells in suspension were collected by centrifugation at 800 g for 5 min. The final pellets were resuspended in 5 ml standard culture medium, plated on 25 cm2 flasks and incubated at 37°C in a humidified 5% CO2 atmosphere. The remaining portions of tissue were repeatedly digested with fresh trypsin six times. After the first passage all the pellets derived from the same tissue were pooled together and an initial single culture was maintained for each tissue, as described above for the eye cell cultures. Clones derived from a single cell were established by seeding an average of 0.5 cells in each well of a 24-well cluster. All the cultures were grown under the same conditions and in the same medium. Once the cells reached 80–100% confluency they were subcultured at a ratio of either 1:40, 1:50 or 1:100. At every passage the number of population doublings was determined based on the cell number.
Immunocytochemistry
Vimentin and cytokeratins were detected immunocytochemically in cultured mouse cells using mouse monoclonal antibodies raised against human proteins, which cross react with mouse. Semiconfluent cells growing on eight-well chamber slides were washed once with serum-free DMEM and fixed in 2% paraformaldehyde in DMEM for 20 min at room temperature. The cells were washed once with PBS, left in 0.1 M glycine for 20 min and washed twice with PBS. Cells were permeabilized in 1% Triton-X100 in PBS for 6 min and washed twice with PBS. Monoclonal anti-vimentin (mouse IgM isotype; Sigma) or monoclonal anti-pan cytokeratins (mouse IgG1 isotype; Sigma) were applied at 1/200 in 0.01% Triton-X100 and incubated overnight at 4°C with gentle shaking. The cells were washed four times in PBS for 5 min. Both anti-mouse IgM–fluorescein isothiocyanate (FITC) conjugate (Sigma) and anti-mouse IgG–TRITC conjugate (Sigma) were applied in a 1/100 dilution in 3% bovine serum albumin in PBS with 0.01% Triton-X100 and incubated for 2 h at room temperature with gentle shaking. Cells were finally washed four times in PBS for 5 min and observed using fluorescence microscopy.
DNA extraction from tissues and cultured cells
Mouse tissue DNA was isolated following phenol/chloroform extraction using standard procedures. Cell culture DNA samples were extracted using a Nucleon DNA extraction kit for blood and tissue culture following the manufacturer’s protocol.
PCR analysis
SP-PCR analyses were performed in a Biometra UNO thermoblock as described previously, with 0.1 µM carrier primer DM-C and using PCR primers DM-C and DM-BR (16). The PCR products were electrophoresed on a 1.25% multipurpose agarose gel (Boehringer Mannheim), transferred onto a nylon membrane (Osmonics), hybridized with a radio labelled double-stranded probe comprising 56 CTG repeats and detected by autoradiography. The PCR products were sized using Kodak Digital Science 1D software.
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ACKNOWLEDGEMENTS |
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We would like to thank the Dynamic Mutation Group at the University of Glasgow and Dr Tetsuo Ashizawa for helpful discussions during the course of this work. We are also grateful to the Biological Services for excellent animal care and assistance with colony maintenance. D.G.M. is a Lister Institute Research Fellow. This study was also supported by grants from Fundação para a Ciência e Tecnologia and Fundação Calouste Gulbenkian (Portugal), to M.G.P., and from the Medical Research Council (UK), to D.G.M.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +44 141 330 6213; Fax: +44 141 330 6871; Email: dmonck@molgen.gla.ac.uk
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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