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G. Amescua, S. Pfahler, E.C. Carlson, V.L. Perez; Exogenous Gene Delivery and Expresion in Human Corneal Stroma Cells Using an Adenoviral Vector . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2606.
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Purpose: To develop a minimally invasive and efficient means of delivering and expressing a gene of interest in the human corneal stroma. Methods: Utilizing the technique of intrastromal injection using 1cc syringe and a 30G needle, 5 x 109 pfu of adenoviral vector expressing an EGFP reporter gene driven by a CMV promoter in a volume of 50 µl was delivered to the corneal stroma of human corneal buttons from the Cleveland Eye Bank. The corneal buttons were then cultured in an incubator at 37 °C and 5%CO2 in RPMI media for a total of 14 days. Fluorecent stereo–micrograph were taken every 24 hrs, using an appropriate filter for EGFP. At 7 days corneal buttons were removed from cultures, DAPI stained and confocal microscopy was performed to generate 3D reconstructions from z–stack images in 1 micron increments. Results:No positive EGFP expression could be detected by fluorescent stereomicroscopy at 24 hrs post–injection; however, expression of the EGFP reporter gene was initially observed at 48 hrs post–injection in 50–60 percent of the total corneal area. The area of expression began to diminish at day 9. By day 14, the corneal area with positive EGFP expression was 19.6 percent. Three–dimensional confocal analysis demonstrated the presence of EGFP was exclusively localized to the stroma Conclusions: Ex vivo transfection of human corneal buttons can be achieved with intrastromal injection of adenoviral vector. This ability of intrastromal injection to deliver an adenoviral construct, may prove useful as a tool to deliver exogenous genes of interest to the corneal stroma.
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