This study was conducted to develop a method of targeted gene transfer by electric pulses to a selected area of corneal stroma in vivo. Plasmid DNA with a green fluorescent protein (GFP) gene under a cytomegalovirus promoter was injected through the corneal pocket into the corneal stroma of the adult Brown Norway rat, and various intensities of electric pulses ranging from 10 to 30 V were delivered to the corneal epithelial side with an electric probe. Direct stereomicroscopy of the fluorescent using realtime imaging was used to determine in vivo gene expression on days 1, 2, 4, 6, 8, 10, 15, and 20 after gene transfer. Transgene expression was detected in the corneal stroma as early as day 1 and until day 15. The most intense expression was noted on days 4 and 6. Gene transfer was most effective using eight electric pulses of 20 V for 50 msec. Histologic study disclosed GFP expression in keratocytes within the targeted area. There was no apparent cell damage in the gene transferred cells. No apparent inflammation was found in the anterior chamber or trabecular cells when electric pulses less than 30 V were used. In summary, the present technique transferred the gene of interest to a highly selected area of corneal stroma with no apparent damage. This method will likely be useful not only for developing gene therapy for corneal diseases but also for corneal research in general.
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