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

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Trinucleotide Repeats

Part of the book series: Methods in Molecular Biology ((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.

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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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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Boonshoft School of Medicine, Wright State University, Dayton, OH, USA

    Rujuta Yashodhan Gadgil, S. Dean Rider Jr., Todd Lewis, Joanna Barthelemy & Michael Leffak

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  1. Rujuta Yashodhan Gadgil
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  2. S. Dean Rider Jr.
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  3. Todd Lewis
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  4. Joanna Barthelemy
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Correspondence to Michael Leffak .

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  1. Department Genomes & Genetics, Institut Pasteur, Paris, France

    Guy-Franck Richard

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Gadgil, R.Y., Rider, S.D., Lewis, T., Barthelemy, J., Leffak, M. (2020). Analysis of Trinucleotide Repeat Stability by Integration at a Chromosomal Ectopic Site. In: Richard, GF. (eds) Trinucleotide Repeats. Methods in Molecular Biology, vol 2056. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9784-8_8

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  • DOI: https://doi.org/10.1007/978-1-4939-9784-8_8

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  • Publisher Name: Humana, New York, NY

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