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Anna Katharina Röpke, Sandra Johnen, Matthias Fuest, Peter Walter, Gabriele Thumann; Genomic Stability in Sleeping Beauty transposon-mediated transfection of RPE cells with the gene for PEDF. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5309.
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The frequent intravitreal injections of VEGF inhibitors necessary to control choroidal neovascularization in exudative AMD patients are accompanied by side effects such as scleral hemorrhage, endophthalmitis, retinal tears, and retinal detachment. To deliver a continuous level of a VEGF inhibitor subretinally, we have transfected RPE cells with the gene for PEDF, an anti-angiogenic and neuroprotective factor, for eventual transplantation to the subretinal space. Transfection was accomplished using the hyperactive Sleeping Beauty (SB100X) transposon system, which results in transgene integration into the host cell’s genome. Here we have analyzed the expression of important genes to insure that the integration of the PEDF gene does not alter the expression of genes critical to RPE function.
Using the SB100X transposon system we transfected ARPE-19 cells with the PEDF gene with 30% efficiency. Quantitative Real-Time PCR was performed to analyze expression levels of genes essential for RPE function, specifically the genes for PEDF, the cytokeratins KRT8 and KRT18, the tight junction protein ZO-1, the angiogenic factor VEGF-A, cathepsin D, the nuclear transcription factor NF-kB, the proto-oncogene c-ABL, the cellular tumor antigen p53, the stress-activated protein kinase JNK1, the visual cycle protein CRALBP, and the apoptosis regulator Bcl-2.
As expected, PEDF gene expression was increased in the transfected cells, which secreted 12.54 ± 0.85 ng/h/105 cells, whereas non-transfected ARPE- 19 cells secreted detectable levels that could not be quantified. No significant changes in expression levels for KRT8, KRT18, ZO-1, VEGF-A, cathepsin D, NF-kB, c-ABL, the cellular tumor antigen p53, JNK1, CRALBP, and Bcl-2 were observed.
Transfection with the hyperactive SB100X transposon system insures that the transgene becomes integrated into the host cell’s genome. Here we have shown that the expression of the PEDF gene is increased without any significant alteration in the expression of a number of genes essential for the normal RPE cell function.
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