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Steven J Fliesler, Pravallika Kotla, Sriganesh Ramachandra Rao, Steven J. Pittler; Lack of overt pathology in a K42E knock-in mouse model of retinitis pigmentosa (RP59). Invest. Ophthalmol. Vis. Sci. 2019;60(9):450.
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© ARVO (1962-2015); The Authors (2016-present)
A K42E point mutation in the dehydrodolichyl diphosphate synthase (DHDDS) gene causes a recessive form of retinitis pigmentosa (RP59). We created a novel mouse model of RP59 to study its underlying pathophysiology mechanism.
CRISPR gRNA was generated to create a double strand break in the DHDDS locus to promote introduction of a single stranded oligodeoxynucleotide carrying the K42E mutation and a second silent DNA polymorphism to eliminate the PAM recognition site required for cleavage by CAS9. Mice (C57BL6/J) heterozygous for the K42E mutation were thus generated and crossed to generate K42E/K42E homozygous mice (confirmed by PCR DNA sequencing). Three potential F0 founder pups were generated from 13 F0 mice, and an F0 founder was verified by DNA sequence analysis. Optical coherence tomography (OCT) was performed at 1 and 3 months (K42E/+, N=6) and 1, 3, and 8 months (K42E/K42E; N=3-7) to assess retinal structure, in comparison with age-matched wild type controls.
Three potential K42E F0 founder pups were generated from 13 F0 mice, and an F0 founder was verified by DNA sequence analysis. OCT analyses were performed on 4 generations of knock-in mice. K42E mutant mouse retinas from all generations exhibited normal cell layer stratification, with no evidence of retinal degeneration or cell loss over the time course examined. OCT results were confirmed by correlative histological analysis.
Despite successful generation of homozygous mutant mice harboring the K42E DHDDS mutation associated with RP59, no retinal degeneration was observed. These results are in contrast to a previous report, using another K42E mouse model (Li Y, et al. ARVO 2014), which found retinal degeneration by 3 postnatal months. The reasons for this discrepancy remain unclear. The RP59 mechanism of retinal degeneration may be more complicated than simple DHDDS loss-of-function.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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