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O.L. Moritz, B.M. Tam; Retinal Degeneration in X. Laevis Induced by Mutant Rhodopsin Transgenes . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3549.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To develop a system for expression of rhodopsin mutants in rod photoreceptor cells and to subsequently study the mechanisms by which these mutations cause photoreceptor cell death leading to retinitis pigmentosa. Previous attempts involved expressing mutant rhodopsin-GFP fusion proteins in transgenic X. laevis rods. This resulted in rapid identification of transgenic animals but low transgene expression levels and no significant retinal degeneration. We will therefore develop an alternative approach that avoids the use of fusion proteins Methods: Using site directed mutagenesis, we have generated a modified X. laevis rhodopsin cDNA whose protein product can be distinguished from wild-type X. laevis rhodopsin by its ability to bind the monoclonal antibody 2B2. We also constructed variants of this sequence that include mutations analogous to P23H and Q344Ter, which cause RP in human patients. We have generated transgenic X. laevis expressing these cDNAs in rod photoreceptors under control of the X. laevis opsin promoter using the method of Kroll and Amaya. Resistance to the antibiotic G418 was used to distinguish transgenic from non-transgenic larvae. Transgenic retinas were analyzed for rhodopsin content by western blot using 2B2, as well as antibodies that recognize both transgenic and endogenous rhodopsin. Transgenic retinas were also analyzed by immunofluorescence microscopy to determine the extent of retinal degeneration, and to localize the transgene products. Results: Using G418 selection we were able to identify transgenic X. laevis expressing non-fluorescent transgenes with a high degree of reliability. G418 selection also greatly reduced the number of transgenic animals with extensive transgene silencing artifacts. Our initial results indicate that by 14 days post-fertilization, high-level expression of the mislocalized Q344Ter variant is strongly correlated with a reduced total rhodopsin content, which in turn is correlated with retinal degeneration. Conclusions: We have developed a novel system for expression of mutant rhodopsins and characterization of their effects on rhodopsin localization and photoreceptor viability. This is the first X. laevis model of retinal degeneration based on a human disease-causing rhodopsin mutant. Future studies will address mechanisms of photoreceptor cell death in these animals.
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