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Orson L. Moritz, Zusheng Zong, Beatrice M. Tam, Cheryl Y. Gregory-Evans, Joanne A. Matsubara, Kevin Gregory-Evans; Screening Potential Drug Therapies For Retinitis Pigmentosa Using Transgenic X. Laevis. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1216.
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The rhodopsin P23H mutation is the most common cause of autosomal dominant RP in North America, and causes instability of opsin in the biosynthetic pathway. Using X. laevis models of retinitis pigmentosa (RP), we have previously demonstrated that retinal degeneration caused by human P23H rhodopsin can be partially rescued by dark rearing due to stabilization of the protein by the 11-cis retinal chromophore; this effect is detectable in tadpoles as soon as two weeks following fertilization. Our purpose is to use this animal model as a medium-throughput screening tool to identify potential drug therapies for RP, including therapies based on pharmacological chaperones, which we hypothesize should demonstrate an effect comparable to dark rearing.
Heterozygous transgenic animals are mated with wildtype animals to generate a population of 50% transgenic animals. Drugs are initially screened for toxicity on X. laevis embryos in order to identify a suitable range for dosing. Mixed groups of transgenic and WT tadpoles are then treated with a half-log dilution series of drug beginning 5 days after fertilization, and concluding 14 days after fertilization, under conditions of either cyclic light or constant darkness. Tadpoles are subsequently processed for PCR-based genotyping and dot blot assay for total rhodopsin. A reduction in total rhodopsin is indicative of retinal degeneration, which can be confirmed by confocal microscopy of the contralateral eye.
As proof-of-principle we tested several small molecule drugs. We found that one researcher was able to complete an entire dose-response experiment with up to 150 individual data points within a four week time frame, including all histology and data analysis. This was significantly faster than testing in mammalian model systems, which took up to 6 months in a comparable experiment involving 125 data points. Because we can monitor the quantity and intracellular localization of transgenic rhodopsin, it is possible to obtain information on both efficacy and mechanism of action.
Transgenic X. laevis provide a mechanistically informative system for rapidly screening potential RP treatments. "Hits" can be further examined in more complex experiments involving mammalian models of RP.
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