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C. Stickelmann, A. Dobias, S. Johnen, G. Thumann; Efficient Genetic Modification of Very Low Numbers of Pigment Epithelial Cells by Non-Viral Transfection Protocols. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4501.
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© ARVO (1962-2015); The Authors (2016-present)
One of the major difficulties in transfection of primary pigment epithelial cells for use in ocular gene therapy is the small number of autologous cells that are obtained either by iridectomy or RPE aspiration. Since transfection protocols require cell numbers in the several hundred thousands, we have investigated the minimum number of freshly isolated and primary cultured IPE and RPE cells that can be efficiently transfected with a plasmid encoding PEDF using non-viral electroporation protocols.
A series of primary cultured RPE and IPE cells ranging from 1 million to 2000 cells were transfected with plasmids encoding PEDF and PEDF-EGFP fusion proteins using different electroporation protocols. Transfection efficiency and stability of PEDF expression were evaluated by fluorescence microscopy and immunoblotting of the secreted Ni-NTA purified PEDF.
Transfection of primary cultured IPE and RPE cells by electroporation was highly efficient in as low as 5000 cells per electroporation. The cells showed good morphological characteristics and expressed recombinant PEDF, even after the cells were passaged, albeit at a reduced level. Low numbers of freshly isolated IPE cells were also successfully transfected, however PEDF expression was transient and was only observed for 8 days.
We have shown that non-viral protocols for transfecting genes in very low numbers of cultured primary IPE and RPE cells is possible, opening the way to be able to transfect autologous pigment epithelial cells with the appropriate genes and to transplant the genetically modified cells to treat retinal degenerations, such as AMD.
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