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Salamander Green Rod/blue cone opsin promoter driven green fluorescent protein expression in transgenic xenopus.

Introduction: Rods and cones are classically described as distinct photoreceptor cells with different receptive characteristics mediated by distinct opsins, but recent studies show 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 work demonstrated photoreceptor expression in transiently transfected Xenopus embryos. We hypothesize that specific promoter elements control the cell-specific expression of this single opsin in both rods and cones.

Methods: The isolated salamander green rod/blue cone promoter was used to replace the CMV promoter in a green fluorescent protein (GFP) reporter plasmid, pEGFPN1(-)SGR/ BCprom. Transgenic Xenopus embryos were created by introducing the plasmid into sperm nuclei and injecting unfertilized eggs. Tadpoles at various stages of m) examined for promotormdevelopment were fixed and cryostat thin sections (14 driven GFP expression using fluorescent microscopy. The presence of transgenic DNA was confirmed by PCR analysis of genomic DNA (gDNA) isolated the tail region of the sectioned Xenopus embryos.

Results: A GFP reporter plasmid construct utilizing the SGR/BC promoter demonstrated GFP expression in photoreceptor cells adjacent to the retinal pigment epithelium. Embryos were confirmed as transgenic with PCR analysis of the Xenopus gDNA for the presence of the GFP gene.

Conclusions: The SGR/BC opsin promoter is capable of driving photoreceptor specific GFP expression in transgenic Xenopus tadpole photoreceptor cells. Future studies will use a series of deletion mutations in the SGR/BC promoter to identify possible regions specific for expression in rods or cones. Jointly supported by NSF/EPSCoR Grant #EPS-0132573 and NIH/BRIN Grant #8-PORR16461A and NSF grant #RUI-344395
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Author:Parker, Ryan; Darden, Alix
Publication:Bulletin of the South Carolina Academy of Science
Date:Jan 1, 2005
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