Knock-out Cell Line Generation

Description

The title of the wider research project is ‘Repeat-induced mutagenesis in Huntington’s disease’ and the overall aim is to understand how expanded CAG/CTG repeats can cause mutagenesis at the Huntington’s disease locus. Huntington’s disease (HD) is a progressive neurodegenerative disease characterised by involuntary movements, apathy, and cognitive decline. Expansion of CAG/CTG repeats within the HTT gene cause HD. These repeats are genetically unstable, leading to dramatic changes in repeat size, biased towards expansion over an individual’s lifetime. Somatic expansion in affected tissues is thought to modify disease presentation and many genes identified in genome-wide association studies play a role in DNA repair and in repeat instability. Repeat instability, defined as gain or loss of repeat units relies on DNA repair processes although the exact mechanisms are still being elucidated. Although several DNA repair components have been implicated in repeat instability, how they work together and how potential sequence context may influence the process is unclear. Part of this project involves investigating the effects of genes associated with DNA repair on repeat instability. 10 different genes of interest have been identified and to investigate their effect on repeat instability, these genes need to be knocked-out in our custom HEK293 cell line. The requirement is for separate knock-out of 10 genes in an immortalised cell line, GFP(CAG)101, which is HEK293-derived. Vincent Dion et al will provide the original cell line as starting material. This material will be mycoplasma tested before being sent.