Purchase this article with an account.
Y. Zhang, T.J. McFarland, B. Appukuttan, S. Chen, D. Burke, J.T. Stout; Efficient and Sustained Long-term Transgene Expresion in Cultured Human and Rat Iris Pigment Epithelium Via Lentiviral Transduction . Invest. Ophthalmol. Vis. Sci. 2003;44(13):441.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: Autologous transplantation of iris pigment epithelium (IPE) cells into the subretinal space can replace lost or dysfunctional retinal pigment epithelium, however IPE cells cannot restore RPE's function completely. Our hypothesis is that IPE cells, transduced with a lentiviral vector containing anti-angiogenic genes, transplanted into the subretinal space can help to treat age-related macular degeneration or other macular diseases. The goal of this study was to observe and test transgene expression in cultured human and rat IPE cells transduced with lentiviral vectors containing the anti-angiogenic genes soluble neuropilin-1 (sNRP1) and soluble kinase insert domain-containing receptor (sKDR). Methods: Human and rat IPE cells were isolated by enzyme-assisted microdissection, and cultured in supplemented Ham's F12 Nutrient Mixture (F12). An eGFP cDNA, sNRP1-FLAG or sKDR-FLAG fusion cDNA was cloned into the plasmid pHR' under the control of the CMV promoter. Replication-defective lentiviral vectors were prepared by three plasmid co-transfection into 293T cells. Human and rat IPE cell cultures were transduced by these lentiviral vectors. Expression of eGFP was analyzed by fluorescence microscopy; RT-PCR utilizing primers specific to the fusion-gene was used to confirm expression within transduced IPE cells. Results: EGFP expression in IPE cells was observed via fluorescent light microscopy, initially at 24 hours after lentiviral transduction. Sustained expression of eGFP was evident for at least 3 months. RT-PCR of transduced IPE cells indicated the presence of sNRP1-FLAG and sKDR-FLAG mRNAs. Conclusions: In vitro, human and rat IPE cells can be efficiently transduced with recombinant lentiviral vectors containing eGFP, sNRP1-FLAG or sKDR-FLAG gene. Genetically modified IPE cells may prove useful in the therapeutic treatment of macular disease.
This PDF is available to Subscribers Only