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DF P Larkin, JC Mc Alister, A Thrasher, RR Ali; Transduction of Stored Murine Corneas with Recombinant Lentivirus Expressing Green Fluorescent Protein (GFP) . Invest. Ophthalmol. Vis. Sci. 2002;43(13):131.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: In order to facilitate the development of gene therapy approaches for improving corneal graft survival in a murine transplantation model, we have optimised ex vivo transduction of mouse corneas using lentiviral vectors so as to preserve tissue function and clarity which is dependent upon storage conditions. Methods: Recombinant lentivirus carrying a GFP reporter gene was prepared using a standard 3-plasmid co-transfection system in 293T cells. Corneas in typical storage media were incubated with 103-109 transduction units for periods of 6-72 hours at either 4o C (normal storage temp) or 37 o C. Specimens were then maintained at 37o C in ex-vivo culture for at least ten days, when transduction efficiency was calculated. Selected specimens were maintained in culture for periods up to 4 months. Results: Incubation of corneas with 106-109 transduction units resulted in efficient transduction of the corneal endothelium (up to 80 %). Transduction efficiency following incubation at 37 o C increased with the period of incubation but plateaued at 48 hours. When corneas were incubated with virus at 4 o C, efficient transduction only occurred following incubation for between 12 and 24 hours. Incubation at 4 o C for 72 hours resulted in minimal transduction. GFP expression was detectable 3 days after incubation with 108 transduction units. Widespread GFP expression was apparent by day 5 following incubation with all but the lowest titres. GFP expression was highest along circumferential stress lines and the cut corneal edge. A subset of corneas showed a marked fall in GFP transduction centrally, after 12 days organ culture while peripheral transduction remained high. Widespread, sustained GFP expression was evident for up to 4 months (the longest time point examined). Conclusion: Efficient and stable transduction of the corneal endothelium with recombinant lentivirus has been demonstrated using protocols for transduction which minimise alterations to normal corneal storage conditions.
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