Sizing, stabilising, and cloning repeat-expansions for gene targeting constructs

Methods. 2021 Jul:191:15-22. doi: 10.1016/j.ymeth.2020.07.007. Epub 2020 Jul 25.


Aberrant microsatellite repeat-expansions at specific loci within the human genome cause several distinct, heritable, and predominantly neurological, disorders. Creating models for these diseases poses a challenge, due to the instability of such repeats in bacterial vectors, especially with large repeat expansions. Designing constructs for more precise genome engineering projects, such as engineering knock-in mice, proves a greater challenge still, since these unstable repeats require numerous cloning steps in order to introduce homology arms or selection cassettes. Here, we report our efforts to clone a large hexanucleotide repeat in the C9orf72 gene, originating from within a BAC construct, derived from a C9orf72-ALS patient. We provide detailed methods for efficient repeat sizing and growth conditions in bacteria to facilitate repeat retention during growth and sub-culturing. We report that sub-cloning into a linear vector dramatically improves stability, but is dependent on the relative orientation of DNA replication through the repeat, consistent with previous studies. We envisage the findings presented here provide a relatively straightforward route to maintaining large-range microsatellite repeat-expansions, for efficient cloning into vectors.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amyotrophic Lateral Sclerosis / genetics
  • Animals
  • C9orf72 Protein / genetics
  • Cloning, Molecular
  • DNA Repeat Expansion*
  • Gene Targeting
  • Humans
  • Mice


  • C9orf72 Protein