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Orson L Moritz, Colette N Chiu, Paloma Stanar, Beatrice M Tam; Electroretinography and difference spectra suggest the presence of alternative pathways for chromophore production in RPE65 knockout Xenopus laevis retina.. Invest. Ophthalmol. Vis. Sci. 2020;61(7):1535.
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© ARVO (1962-2015); The Authors (2016-present)
RPE65 is an isomerohydrolase that converts all-trans-retinyl ester to 11-cis-retinol, which is then oxidized into 11-cis-retinal, the ligand for rod and cone opsins, by downstream enzymes. Studies in Rpe65-/- mice have shown an almost complete depletion of 11-cis-retinal in photoreceptors and an associated retinal degeneration with rapid cone degeneration. However, alternative pathways for production of chromophore have been proposed, which may function to provide chromophore for cones. We have generated RPE65 knockout X. laevis; here we examine the possibility of RPE65-independent chromophore production in X. laevis retina.
RPE65 knockouts were generated using CRISPR/Cas9 technology by targeting exon 7 of both the rpe65.L and rpe65.S genes in single-cell X. laevis embryos. Animals were analyzed at (i) 14 days post fertilization by immunohistochemistry, DNA sequencing, and western blotting; (ii) 6 weeks post fertilization by scotopic, photopic, and flicker electroretinagram, DNA sequencing, and immunohistochemistry; (iii) 4 months post fertilization by difference spectra obtained from retinas isolated from dark adapted animals.
Sequencing analysis indicated that ~25% of F1 inbred animals retained one WT rpe65.S allele, while all analyzed animals were edited at the rpe65.L gene. No RPE65 protein could be detected in edited animals by western blot regardless of genotype. Immunohistochemistry showed markedly reduced RPE65 protein expression with no retinal degeneration at any age; cones were present. No rhodopsin pigment was apparent in difference spectra, although other hydroxylamine-sensitive peaks were present. Scotopic ERG was abolished, while photopic ERG a-wave and b-wave amplitude were reduced. ERG responses to bright red flashes increased significantly in the presence of a blue background.
Using CRISPR/Cas9 technology, we have generated RPE65 knockout X. laevis, and transmitted the resulting genotype and phenotype to the F1 generation. These animals are similar to mammalian RPE65 knockouts in that they have minimal rhodopsin chromophore. However, the cones do not degenerate, and other photopigments may be present at sufficient levels to drive cone responses. Increased ERG responses in the presence of blue light are consistent with proposed photoreversal-based mechanisms for chromophore regeneration.
This is a 2020 ARVO Annual Meeting abstract.
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