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

ABSTRACT: 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 gr

SUBMITTER: Nair RR 

PROVIDER: S-EPMC8215685 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature