Mouse tissue culture models of unstable triplet repeats: in vitro selection for larger alleles, mutational expansion bias and tissue specificity, butno association with cell division rates

The expansion of CAG(.)CTG trinucleotide repeats has been associated with a n increasing number of human diseases, Once into the expanded disease-assoc iated range, the repeats become dramatically unstable in the germline and a lso throughout the soma, Instability is expansion-biased, contributing towa rds the unusual genetics, and most likely the tissue-specificity and progre ssive nature of the symptoms. Such expansions constitute a unique form of d ynamic mutation whose mechanism is poorly understood. It is generally assum ed 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 th at reconstitute the dynamic nature of somatic mosaicism observed in humans. We have now used tissues from these mice to establish in vitro cell cultur es. Monitoring of repeat stability in these cells has revealed the progress ive 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 cultu res and frequent additional selective sweeps at later stages. The highest l evels of instability were observed in cultured kidney cells, whereas the tr ansgene remained relatively stable in eye cells and very stable in lung cel ls, paralleling the previous in vivo observations. No correlation between r epeat instability and the cell proliferation rate was found, rejecting a si mple association between length change mutations and cell division, and con firming a role for additional cell-type specific factors.