Determining the molecular mechanisms that contribute to trinucleotide repeat (TNR) expansions is essential to understanding the origin of genetically inherited diseases, such as Huntington’s disease, and to inform efforts in developing therapeutic treatments. As one resource to probe the mechanisms of TNR expansions, we describe an expansion assay in human tissue culture cells. The cell line SVG-A, derived from human astrocytes, has the important property of supporting expansions in culture, unlike many cell lines derived from patients. SVG-A cells are also amenable to standard genetic and biochemical techniques such as siRNA, CRISPR-Cas9 and enzymatic inhibitors. This combination of features allows for mechanistic studies of TNR expansions, using the quantitative genetic assay described here as a readout. The SVG-A assay has correctly identified key proteins that drive expansions and it has facilitated testing of enzymatic inhibitors that suppress expansions as potential therapeutics. This chapter describes how repeat expansions are detected, visualized, and quantified.
Trinucleotide repeats Expansion Genome instability DNA mutagenesis Huntington’s disease Reporter construct Polymerase chain reaction Astrocytes Human cells
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This work was supported by Government of Ireland Postdoctoral Fellowship GOIPD/2018/291 from the Irish Research Council (to Gregory M. Williams) and by Science Foundation Ireland grant 16/BBSRC/3395 (to Robert S. Lahue).
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