Construction of cell-culture based assay system to understand CGG repeat instability in Fragile X syndrome

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The human Fragile X mental retardation 1 (FMR1) gene, which contains CGG-trinucleotide repeat in its 5'UTR region, is responsible gene for Fragile X syndrome (FXS). The number of CGG-repeat is about 50 to 200 in carriers (so-called 'premutation allele') and more in patients. CGG-repeat becomes unstable and in most cases expands upon maternal inheritance of premutation allele to children. However, the mechanism underling CGG-repeat instability is unknown to date. In the present study, toward elucidation the molecular and cellular mechanism in CGG-repeat instability, we tried to establish a cell culture-based system that enables to reproduce CGG-repeat instability in vitro. We applied chromosomal engineering so that we could clone and handle CGG-repeat tract as a chromosomal domain stably. We successfully cloned natural and artificial chromosomes that contain varying size of CGG-repeats in rodent cell lines.