June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Rpe65 knockout prevents T4K rhodopsin-induced retinal degeneration in a transgenic X. laevis model of retinitis pigmentosa
Author Affiliations & Notes
  • Paloma Stanar
    Ophthalmology and Visual Sciences, University of British Columbia, Vancouver, British Columbia, Canada
  • Beatrice M Tam
    Ophthalmology and Visual Sciences, University of British Columbia, Vancouver, British Columbia, Canada
  • Ruanne Vent-Schmidt
    Ophthalmology and Visual Sciences, University of British Columbia, Vancouver, British Columbia, Canada
  • Orson L Moritz
    Ophthalmology and Visual Sciences, University of British Columbia, Vancouver, British Columbia, Canada
  • Footnotes
    Commercial Relationships   Paloma Stanar, None; Beatrice Tam, None; Ruanne Vent-Schmidt, None; Orson Moritz, None
  • Footnotes
    Support  Foundation Fighting Blindness (Canada), National Sciences and Engineering Research Council of Canada (NSERC) RGPIN-2015-04326
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 4548. doi:
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    • Get Citation

      Paloma Stanar, Beatrice M Tam, Ruanne Vent-Schmidt, Orson L Moritz; Rpe65 knockout prevents T4K rhodopsin-induced retinal degeneration in a transgenic X. laevis model of retinitis pigmentosa. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4548.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Mutations in rhodopsin a commonly implicated cause of retinitis pigmentosa (RP), yet the mechanism underlying the resulting death of rods remains unclear. In transgenic X. laevis models of RP, we previously examined retinal degeneration (RD) caused by the RP-causing rhodopsin mutation T4K, and determined that light is required to induce RD, while vitamin A deficiency prevents RD. This suggests not only a requirement for the chromophore and photoactivated mutant rhodopsin, but also the possibility of a genetic interaction between T4K rhodopsin and rpe65, the gene encoding the isomerohydrolase responsible for chromophore regeneration. Here we tested chromophore dependence of RD caused by T4K rhodopsin via knockout (KO) of the genes encoding Rpe65 using CRISPR/Cas9 technology in X. laevis.

Methods : Cas9, eGFP, and sgRNA targeting sites within exon 7 of the X. laevis rpe65 genes were transcribed in vitro and injected into fertilized eggs obtained from transgenic X. laevis females expressing T4K rhodopsin crossed with wildtype males. Cas9-injected embryos were used as controls. 14 day-old tadpoles were sacrificed and examined by DNA sequencing. Solubilized eyes were assessed by western and dot blot for Rpe65 and rod opsin, while contralateral eyes were examined by immunolabeling for confocal microscopy. Visual function of KOs was assessed by electroretinography (ERG).

Results : Direct sequencing of PCR products indicated extensive editing of rpe65. KO animals showed loss of Rpe65 immunolabeling in cryosections, and minimal or no Rpe65 protein on western blot and abnormal ERG relative to controls. KO prevented RD in animals expressing T4K rhodopsin, but had minimal effects on non-transgenic retinas. Dot blot analysis for total rod opsin demonstrated a dramatic genetic interaction between the T4K rhodopsin transgene and rpe65.

Conclusions : Our findings confirm the existence of a mechanism of RD induced by T4K rhodopsin in which the presence of light and bound chromophore exacerbates rod death. Our results indicate that vitamin A therapy is contraindicated for RP patients with the T4K rhodopsin genotype. Furthermore, our results demonstrate rapid assessment of genetic interactions in X. laevis models of RP via gene KO in F0 animals. We are currently examining chromophore dependence in other X. laevis RP models in order to identify genotype-dependent differences in RD mechanisms.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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