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R. Parker, A. Darden; Specificity of the Salamander Green Rod–Blue Cone Opsin Promoter in Transgenic Xenopus Embryos . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3079.
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© ARVO (1962-2015); The Authors (2016-present)
Introduction: Rods and cones are classically described as distinct photoreceptor cells with different receptive characteristics mediated by distinct opsins. Recent studies have shown that salamander blue–sensitive cones and green rods both express an identical opsin. Because this opsin is expressed in two distinct cell types, we believe there are rod and cone specific promoter elements driving expression in each cell. The promoter for the salamander green rod–blue cone (SGR/BC) opsin has been sequenced, and our prior studies demonstrated photoreceptor expression in transiently transfected Xenopus embryos. We hypothesize that in transgenic frogs, green fluorescent protein (GFP) under the control of the SGR/BC promoter will be expressed in both the green rods and blue cones. Methods: The isolated salamander green rod/blue cone promoter was used to replace the CMV promoter in a GFP reporter plasmid, pEGFPN1(–)SGR/BCprom. Transgenic Xenopus embryos were created by introducing the plasmid into sperm nuclei and injecting unfertilized eggs. After developing to stage 45, transgenic embryos were identified with PCR analysis of tail sections. The confirmed transgenic embryos were fixed and cryostat thin sections (14µ m) were examined for promoter driven GFP expression in photoreceptors with fluorescent microscopy. Results: PCR analysis confirmed the presence of transgenic DNA containing the GFP gene, and microscopy was performed on confirmed transgenic embryos. The GFP reporter plasmid construct under the control of the SGR/BC promoter demonstrated GFP expression in photoreceptor cells. Conclusions: The SGR/BC promoter is capable of driving photoreceptor specific GFP expression in transgenic Xenopus embryos. The efficacy of the SGR/BC promoter in transgenic Xenopus embryos will provide an appropriate model for future studies that will use a series of deletion mutations to identify possible regions that drive selective expression in either rods or cones
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