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Kelly Ziaka, Kwan-Leong Hau, Dimitra Athanasiou, Rosellina Guarascio, Mònica Aguilà, James Bellingham, Smriti A. Agrawal, Yumei Li, Rui Chen, Michael E Cheetham; Development of knock in (KI) mouse models of rhodopsin retinitis pigmentosa . Invest. Ophthalmol. Vis. Sci. 2019;60(9):441. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Inherited mutations in the light sensitive protein rhodopsin are the most common cause of autosomal dominant Retinitis Pigmentosa (RP). Rhodopsin mutations can be grouped in distinct classes based on their biochemical and cellular properties; however, many variants have not been studied in detail in vivo due to the lack of appropriate animal models. Here, we have created knock-in mice that genetically represent the M39R, T58R, R135W, and P347L mutations that are associated with dominant RP in individuals in the UK.
CRISPR/CAS9 gene editing was used for the creation of the M39R, T58R, R135W, and P347L knock in mice. Knock in production was achieved by the coinjection of Cas9 and gRNA with single stranded (ss) DNA repair template, which was designed to induce the desired genetic change, for homology directed repair (HDR). Sanger sequencing was used to identify founder mice (F0). Founders were then crossed with C57BL/6J mice. In addition, a new restriction enzyme site was created in the targeted allele for the screening of positive animals on F1 generation.
We successfully created genetically altered founder mice carrying M39R, T58R, R135W, and P347L rhodopsin mutations that cause RP. After the CRISPR/Cas9 editing of the embryos, F0 animals were outcrossed with control mice and inbred to produce, both homozygous and heterozygous animals on a C57BL/6J background. Mouse colonies were successfully expanded. The gene editing showed no deleterious effects on mouse health. Experimental animals are now subject to detailed investigation of visual function and retinal morphology.
Generation of these new M39R, T58R, R135W, and P347L mouse models that recapitulate RP mutations at the correct gene dosage will enable the detailed investigation of the these rhodopsin variants in vivo. These new KI models will also provide insights about the disease mechanisms and facilitate future testing of potential new therapies for rhodopsin RP.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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