Generation of C9orf72h370 mice, an intron 1 humanised C9orf72 repeat-expansion knock-in model
Authors list
Remya R Nair Mireia Carcolé David Thompson Charlotte Tibbit Ross McLeod Alexander Cammack Tatiana Jakubcova Daniel Biggs Matthew Wyles Matthew Parker Adam Caulder Lydia Teboul Chloe L Fisher-Ward Ali R Awan Michael Flower Ben Davies Adrian M Isaacs Elizabeth MC Fisher Thomas J CunninghamThis article is a preprint. Preprints have not been peer-reviewed.
You can read more about preprints.
Abstract
An autosomal dominant GGGGCC repeat expansion in intron 1 of the C9orf72 gene is the most common genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here, we set out to engineer a gene targeted mouse model harbouring a pathogenic length humanised C9orf72 repeat expansion allele, in order to model pathological mechanisms in a physiological context. In human disease, pathogenic repeats typically range from the hundreds to thousands of units in length, representing a considerable challenge for cellular and in vivo model generation given the instability of GC rich and repetitive DNA sequences during molecular cloning. To overcome this challenge, we developed new methodology to synthetically and iteratively build pure GGGGCC repeats within a linear vector system, which we then seamlessly and scarlessly embedded within the native human genomic sequence. This created a gene targeting DNA vector for homologous recombination of the human sequence in mouse embryonic stem cells. We used this novel targeting vector to generate a new gene targeted mouse allele, C9orf72h370, that for the first time has mouse C9orf72 intron 1 scarlessly replaced with human intron 1 including a pure (GGGGCC)370 hexanucleotide repeat expansion. We confirm that the mouse model expresses human intron 1-derived RNA and produces dipeptide repeat proteins derived from the GGGGCC repeat expansion. We now provide this model as a new freely available resource for the field. In addition, we demonstrate the utility of our cloning method for engineering diverse repeat expansion sequences for modelling other disorders, such as Fragile X Syndrome.
Details
Archive
bioRxiv
Available online
Publication date
Keywords
Type of publication
