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A. Liu, C. Kim, L. A. Oliveira, M. I. Rosenblatt; Gene Transfer to Primary Corneal Epithelial Cells via Lentiviral Vectors. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3759.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate the efficiency of heterologous genes transfer to cultured corneal epithelial cells in vitro.
Freshly enucleated rabbit corneoscleral tissue was used to obtain corneal epithelial cell suspensions via double enzymatic digestion. Cells were plated at a density of 2x104/cm2 and allowed to grow to 70-80% confluence prior to transduction. Gene transfer was monitored using fluorescence microscopy and fluorescence activated cell sorting (FACS). We evaluated the transduction efficiency (TE) of different lentivirus concentrations as well as the expression of GFP in transduced cells over time and over multiple passages. Early passage cells were dual sorted by FACS for GFP expression as well as Hoechst dye exclusion to evaluate the transduction efficiency of immature side-population cells.
GFP expressing lentiviral vectors (titers of 7.5 X 106 cfu/ml) were able to effectively transduce rabbit primary epithelial cell culture ex vitro. Live cell imaging post-transduction demonstrated GFP-positive cells with normal epithelial cell morphology and growth. The TE was highest at the 3rd post-transduction day and tended to stabilize after that day. The number of transduced cells was dose-dependent, and at the highest lentivirus concentrations approached 77.9%. After passaging, more mature cells were less easily transduced. When double sorted by FACS to isolate both GFP positive and side population cells, transduced side population cells were identified and the TE was similar to that observed for the entire corneal cell population.
Lentiviral vectors can effectively transfer heterologous genes to cultured corneal epithelial cells in vitro. Genes are stably expressed over time, transferred in a dose-dependence fashion, and can be transferred to mature corneal cells as well as putative stem cells.
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