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Experimental System to Study Instability of (CGG)n Repeats in Cultured Mammalian Cells

  • Artem V. Kononenko
  • Thomas Ebersole
  • Sergei M. MirkinEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2056)

Abstract

Expansions of simple trinucleotide repeats, such as (CGG)n, (CAG)n or (GAA)n, are responsible for more than 40 hereditary disorders in humans including fragile X syndrome, Huntington’s disease, myotonic dystrophy, and Friedreich’s ataxia. While the mechanisms of repeat expansions were intensively studied for over two decades, the final picture has yet to emerge. It was important, therefore, to develop a mammalian experimental system for studying repeat instability, which would recapitulate repeat instability observed in human pedigrees. Here, we describe a genetically tractable experimental system to study the instability of (CGG)n repeats in cultured mammalian cells (Kononenko et al., Nat Struct Mol Biol 25:669–676, 2018). It is based on a selectable cassette carrying the HyTK gene under the control of the FMR1 promoter with carrier-size (CGG)n repeats in its 5′ UTR, which was integrated into the unique RL5 site in murine erythroid leukemia cells. Expansions of these repeats and/or repeat-induced mutagenesis shut down the reporter, which results in the accumulation of ganciclovir-resistance cells. This system is useful for understanding the genetic controls of repeat instability in mammalian cells. In the long run, it can be adjusted to screen for drugs that either alleviate repeat expansions or reactivate the FMR1 promoter.

Keywords

Trinucleotide repeats Repeat expansions Repeat-induced mutagenesis Fragile X syndrome Mammalian cell culture RNA interference 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Artem V. Kononenko
    • 1
  • Thomas Ebersole
    • 1
  • Sergei M. Mirkin
    • 1
    Email author
  1. 1.Department of BiologyTufts UniversityMedfordUSA

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