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S. Kachi, N. Esumi, D.J. Zack, P.A. Campochiaro; In vivo Electroporation of Plasmid DNA: A Tool for Promoter Analysis in RPE Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5197.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To explore the feasibility of using in vivo electroporation of plasmid DNA as a quantitative method for promoter analysis in RPE cells. Methods: Adult mice were given a subretinal injection of plasmids containing the firefly luciferase gene driven by different fragments of the 5’–upstream region of the vitelliform macular dystrophy 2 (VMD2) gene (pGL2–VMD2), followed by electroporation. A plasmid containing the Renilla luciferase gene driven by the CMV promoter (pRL–CMV) was co–injected to normalize the firefly luciferase activity. Mouse eyes were harvested on day 3, and dual luciferase assays were performed on posterior eyecup lysates. These results were interpreted to reflect RPE expression because prior studies had shown that similar constructs with a lacZ reporter lead to RPE–specific expression. Results: Normalized luciferase assays with –585/+38, –253/+38, and –104/+38bp VMD2 plasmids yielded relative activities of 905, 405, and 32, respectively, relative to empty pGL2 vector, suggesting the presence of a strong positive regulatory element(s) between –253 and –104 bp. In addition, when a fragment from intron 1 was combined with the –585/+38bp fragment, activity was reduced by 60%. To narrow down the region responsible for this repression, deletion analysis was performed and exclusion of a 300 bp region near the 5’–end of intron 1 eliminated the repression, whereas the other deletions tested had minimal if any effect. These data correlate well overall with previous studies in transgenic mice carrying VMD2 promoter–LacZ constructs as well as transient transfection assays in D407 human RPE cells. Conclusions: Subretinal injection of plasmid DNA followed by electroporation provides a useful new tool for promoter analysis in RPE cells. Using this method, we have identified repressor activity in the 300bp region near the 5’–end of intron 1 of the VMD2 gene.
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