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

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


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.


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


  1. 1.
    Paulson H (2018) Repeat expansion diseases. Handb Clin Neurol 147:105–123CrossRefGoogle Scholar
  2. 2.
    Mirkin SM (2007) Expandable DNA repeats and human disease. Nature 447:932–940CrossRefGoogle Scholar
  3. 3.
    Usdin K, NCM H, Freudenreich CH (2015) Repeat instability during DNA repair: insights from model systems. Crit Rev Biochem Mol Biol 50:142–167CrossRefGoogle Scholar
  4. 4.
    Jin P, Warren ST (2000) Understanding the molecular basis of fragile X syndrome. Hum Mol Genet 9:901–908CrossRefGoogle Scholar
  5. 5.
    Imbert G, Feng Y, Nelson DL et al (1998) FMR1 and mutations in fragile X syndrome: molecular biology, biochemistry and genetics. In: Wells RD, Warren ST (eds) Genetics instabilities and hereditary neurological disorders. Academic Press, San Diego, pp 27–53Google Scholar
  6. 6.
    Kunst CB, Warren ST (1994) Cryptic and polar variation of the fragile X repeat could result in predisposing normal alleles. Cell 77:853–861CrossRefGoogle Scholar
  7. 7.
    Hagerman RJ, Hagerman PJ (2002) The fragile X premutation: into the phenotypic fold. Curr Opin Genet Dev 12(3):278–283CrossRefGoogle Scholar
  8. 8.
    Sherman SL (2000) Premature ovarian failure among fragile X premutation carriers: parent-of-origin effect? Am J Hum Genet 67:11–13CrossRefGoogle Scholar
  9. 9.
    Man L, Lekovich J, Rosenwaks Z et al (2017) Fragile X-associated diminished ovarian reserve and primary ovarian insufficiency from molecular mechanisms to clinical manifestations. Front Mol Neurosci 10:290CrossRefGoogle Scholar
  10. 10.
    Kenneson A, Zhang F, Hagedorn CH et al (2001) Reduced FMRP and increased FMR1 transcription is proportionally associated with CGG repeat number in intermediate-length and premutation carriers. Hum Mol Genet 10:1449–1454CrossRefGoogle Scholar
  11. 11.
    Raca G, Siyanova EY, Mirkin SM (2000) Janus effects of premutation size CGG repeats on gene expression. Am J Hum Genet 67:366Google Scholar
  12. 12.
    Tassone F, Hagerman RJ, Taylor AK et al (2000) Elevated levels of FMR1 mRNA in carrier males: a new mechanism of involvement in the fragile-X syndrome. Am J Hum Genet 66:6–15CrossRefGoogle Scholar
  13. 13.
    El-Osta A (2002) FMR1 silencing and the signals to chromatin: a unified model of transcriptional regulation. Biochem Biophys Res Commun 295(3):575–581CrossRefGoogle Scholar
  14. 14.
    Hansen RS, Canfield TK, Lamb MM et al (1993) Association of fragile X syndrome with delayed replication of the FMR1 gene. Cell 73:1403–1409CrossRefGoogle Scholar
  15. 15.
    Kononenko AV, Ebersole T, Vasquez KM et al (2018) Mechanisms of genetic instability caused by (CGG)n repeats in an experimental mammalian system. Nat Struct Mol Biol 25:669–676CrossRefGoogle Scholar
  16. 16.
    Lupton SD, Brunton LL, Kalberg VA et al (1991) Dominant positive and negative selection using a hygromycin phosphotransferase-thymidine kinase fusion gene. Mol Cell Biol 11:3374–3378CrossRefGoogle Scholar
  17. 17.
    Feng YQ, Lorincz MC, Fiering S et al (2001) Position effects are influenced by the orientation of a transgene with respect to flanking chromatin. Mol Cell Biol 21:298–309CrossRefGoogle Scholar
  18. 18.
    Ebersole T, Kim J-H, Samoshkin A et al (2011) tRNA genes protect a reporter gene from epigenetic silencing in mouse cells. Cell Cycle (Georgetown, Tex) 10:2779–2791CrossRefGoogle Scholar
  19. 19.
    Shah KA, Mirkin SM (2015) The hidden side of unstable DNA repeats: mutagenesis at a distance. DNA Repair 32:106–112CrossRefGoogle Scholar
  20. 20.
    Shah KA, Shishkin AA, Voineagu I et al (2012) Role of DNA polymerases in repeat-mediated genome instability. Cell Rep 2:1088–1095CrossRefGoogle Scholar
  21. 21.
    Anand RP, Lovett ST, Haber JE (2013) Break-induced DNA replication. Cold Spring Harb Perspect Biol 5:a010397CrossRefGoogle Scholar
  22. 22.
    Malkova A, Ira G (2013) Break-induced replication: functions and molecular mechanism. Curr Opin Genet Dev 23:271–279CrossRefGoogle Scholar
  23. 23.
    Costantino L, Sotiriou SK, Rantala JK et al (2014) Break-induced replication repair of damaged forks induces genomic duplications in human cells. Science 343:88–91CrossRefGoogle Scholar
  24. 24.
    Sotiriou SK, Kamileri I, Lugli N et al (2016) Mammalian RAD52 functions in break-induced replication repair of collapsed DNA replication forks. Mol Cell 64:1127–1134CrossRefGoogle Scholar
  25. 25.
    Pasero P, Vindigni A (2017) Nucleases acting at stalled forks: how to reboot the replication program with a few shortcuts. Annu Rev Genet 51:477–499CrossRefGoogle Scholar
  26. 26.
    Kramara J, Osia B, Malkova A (2017) Break-induced replication: an unhealthy choice for stress relief? Nat Struct Mol Biol 24:11–12CrossRefGoogle Scholar

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

Personalised recommendations