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H Yang, E Chaum; Expression of Human IGF-1 Fusion Gene Vectors in Retinal Pigment Epithelial Cells Following In Vitro Gene Transfer . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4630.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The cytokine insulin-like growth factor-1 (IGF-1) is highly regulated in retinal pigment epithelial (RPE) cells and likely plays an important role in regulating the growth of RPE cells and their response to injury. It is possible to induce the expression of IGF-1 in cells by directed transfer of IGF-1 genes and by doing so, potentially modify the RPE cell phenotype. In this study, we transfected RPE cells in vitro using fusion gene vectors encoding modified IGF-1 genes which permitted us to distinguish between endogenous and transgenic IGF-1 expression. Methods: We constructed two modified IGF-1 fusion genes containing a cDNA copy of the hIGF-1 gene linked to marker sequences. Plasmids pEGFP:IGF-1 and pcDNA:IGF-1 link the hIGF-1 gene to green fluorescent protein and His6 epitope sequences respectively. Transfected RPE cells were cultured in selective medium and cloned. Expression of fusion gene specific transcripts was confirmed by RT-PCR analysis. IGF-1 fusion proteins were detected by Western blotting using antibodies to IGF-1 and GFP. The His6-tagged IGF-1 fusion protein was purified by immunoprecipitation and nickel column chromatography. Results: In both pEGFP:IGF-1 and pcDNA:IGF-1 transfected RPE clones, fusion gene transcripts were detected at high levels by RT-PCR. In cells with high levels of pEGFP:IGF-1 expression, clones were detected by fluorescence microscopy. Fusion proteins encoded by both vectors were identified by Western blotting. Our studies show that transfected IGF-1 fusion genes are expressed in transduced RPE cells. Conclusion: Modified IGF-1 fusion genes were introduced into RPE cells and were highly expressed in transduced clones. These vectors are molecular tools which may be used to modify the expression pattern of IGF-1 in recipient cells using gene transfer.
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