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H. Dannowski, J. Bednarz, R. Reszka, K. Engelmann, U. Pleyer; Lipid-mediated Gene Transfer of EGFP into Human Corneal Endothelial Cells (HCEC) . Invest. Ophthalmol. Vis. Sci. 2003;44(13):845.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Although in generally not as efficient as viral vectors, nonviral systems have potential advantages in gene transfer. These include a lack of immunogenicity and commercially availability. The purpose of this study was to examine efficiency and toxicity of different nonviral transfection reagents by FACS-analysis after transfection of human corneal endothelial cells (HCEC). Methods: An immortalized cell line of HCEC was cultured and transfected with a plasmid coding for EGFP. Various transfection reagents including DAC30, Fugene 6 (Roche), Lipofectamine, Lipofectin, CellFectin, DMRIE-C (Gibco), Effectene, SuperFect (Qiagen) were studied for gene transfer. HCEC were seeded at 4x104 cells/well in 24 well plates, transfected and quantified 24 hours after transfection. Untransfected HCEC served as controls. Efficiency was determined by EGFP-Fluorescence measurement and toxicity by propidium iodide (PI) staining. Results: Gene transfer efficiency and toxicity of the transfection reagents are strongly dependent on the lipid:DNA ratios and concentrations as indicated by EGFP expression and PI staining. For every transfecting agent conditions need to be optimised. In these experiments efficiencies of about 10% with toxicities of about 3% were decided to be useful in transfecting HCEC. The agents are listed here in order of their applicability: Lipofectin > DMRIE-C > FuGene6, Effectene > DAC30 > CellFectin > Lipofectamine > Superfect. Conclusions: Transfection with EGFP and subsequently PI staining is a feasible way for selection of optimal lipid:DNA ratios for the highest potential efficiency and lowest toxicity of different nonviral transfection reagents. Lipid-mediated gene transfer is a promising approach to target corneal endothelial cells. Careful examination should be performed for optimal results with nonviral transfection reagents to use all their advantages.
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