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Analysis of Trinucleotide Repeat Stability by Integration at a Chromosomal Ectopic Site

  • Rujuta Yashodhan Gadgil
  • S. Dean RiderJr.
  • Todd Lewis
  • Joanna Barthelemy
  • Michael LeffakEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2056)

Abstract

Expansions of CNG microsatellite tracts are responsible for several neurodegenerative diseases, including myotonic dystrophy type 1, Huntington disease, and spinocerebellar ataxia type 8. Here we show that expanded (CNG)n repeats are susceptible not only to expansions and contractions, but are prone to DNA double strand breaks following replication stress. We describe a general strategy for the construction of clonal cell lines containing CNG repeats of various lengths, in which the microsatellites are integrated using the yeast FLP recombinase at a single ectopic recombination acceptor site in the HeLa genome. We illustrate two types of (CTG/CAG) cell lines, one of which contains dual fluorescent marker genes flanking the (CTG/CAG) repeat, and one which does not. We show that long CNG repeats are prone to DNA double strand breaks (DSBs) upon exposure of these cell lines to prolonged replication stress.

Keywords

Microsatellite Trinucleotide repeat FLP recombinase Chromosome ectopic site Ganciclovir Polymerase chain reaction (PCR) Small-pool PCR DNA double strand break Flow cytometry 

Notes

Acknowledgments

This work was supported by NIH/NIGMS grant GM122976 to M.L., and by the WSU BMS Ph.D. program (T.L., J.B.). We thank David Hitch and French Damewood IV for comments on the manuscript.

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

Authors and Affiliations

  • Rujuta Yashodhan Gadgil
    • 1
  • S. Dean RiderJr.
    • 1
  • Todd Lewis
    • 1
  • Joanna Barthelemy
    • 1
  • Michael Leffak
    • 1
    Email author
  1. 1.Department of Biochemistry and Molecular Biology, Boonshoft School of MedicineWright State UniversityDaytonUSA

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