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A. Cho, T.J. McFarland, Y. Zhang, B. Appukuttan, J.T. Stout; Production of a Lentiviral Vector Containing the Soluble Form of the Receptor for Advanced Glycosylation End Products: A Potential Inhibitor of Diabetic Retinopathy . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3198.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The binding of advanced glycosylation end products (AGE) to the receptor for AGE (RAGE) is implicated in the pathogenesis of diabetic vasculopathy. The soluble isoform of RAGE (sRAGE) is a known protective agent against vascular hyperpermeability. We have developed a recombinant lentiviral vector capable of efficiently delivering the transgene sRAGE to cells. We aim to test the efficacy of sRAGE in its ability to decrease AGE induced toxicity observed in pericytes, a disease process associated with diabetic retinopathy. Methods: The full–length cDNA of RAGE was modified by PCR to the soluble form using a 60 bp reverse primer coding for the 3’ soluble portion of sRAGE. Amplified sRAGE cDNA was cloned into the lentiviral plasmid pHR’–IRES–eGFP under the control of the CMV promoter. Replication–defective sRAGE lentivirus was produced by cotransfection into 293T cells. Expression of sRAGE in human microvascular endothelial cells transduced with this virus was analyzed by RT–PCR. Currently AGE products are being produced in vitro. Briefly, 50 mg/ml of recombinant human serum albumin is incubated with 50mM glucose in the presence of antibiotics and PMSF for 6–8 weeks at 37° C. Results: The cDNA coding sRAGE was successfully cloned into a pHR’CMV–IRES–eGFP vector. Replication defective virus has been made and AGE production is underway. Conclusions: A lentiviral reagent containing sRAGE has been produced. This reagent may prove useful in mitigating pericyte loss and vascular hyperpermeability and, thus, may be useful in the prevention/treatment of diabetic retinopathy. To test this theory we are currently examining the toxicity effects of AGE on both sRAGE transduced and nontransduced pericytes.
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