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Nina Harmening, Sandra Johnen, Jeff Bierwagen, Peter Walter, Zsuzsanna Izsvak, Gabriele Thumann; Doxycycline-Dependent Regulation of PEDF Gene Expression in Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):28.
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© ARVO (1962-2015); The Authors (2016-present)
Transplantation of pigment epithelial cells expressing recombinant pigment epithelium-derived factor (PEDF) into the subretinal space is a promising treatment modality for AMD. Using the hyperactive Sleeping Beauty (SB100X) transposase system, our laboratory has achieved the stable, non-virally-mediated transfection of pigment epithelial cells with the PEDF gene. To avoid undesirable side effects as well as for appropriate control of neovascularization in diseases such as AMD, it is preferable to regulate the level and the timing of PEDF expression. For such purpose we established a doxycycline-inducible system that provides a valuable tool to allow for the quantitative and temporal modulation of PEDF expression by transfected cells.
Transposon plasmids comprising a Tet-inducible promoter directing the expression of a transgene and the reverse tetracycline transactivator (rtTA) were constructed in a way that the transactivator is activated by doxycycline - a derivate of tetracycline - which binds to the promoter to induce gene transcription. Using microporation, ARPE-19 cells were transfected with a plasmid encoding the Tet-inducible promoter, rtTA, and EGFP or PEDF-EGFP as well as a plasmid encoding the SB100X transposase. Cells were incubated with different doxycycline concentrations to evaluate effect on cell viability and dose-dependency of EGFP and PEDF expression.
Using the SB transposase system, ARPE-19 cells were stably transfected with efficiencies close to 100%. The use of a doxycycline responsive element in the transposons allows for the external induction of gene expression. Due to the leakiness of the Tet-inducible promoter in the absence of doxycycline, PEDF transfected cells secrete a small amount of PEDF. In the presence of 10 to 1000 ng/ml doxycycline transfected ARPE-19 cells respond with an approximately 6-fold increase in PEDF secretion. However, doses of 10 µg/ml doxycycline or higher are toxic to ARPE-19 cells resulting in a parallel decrease in PEDF secretion.
The inclusion of rtTA into the PEDF encoding plasmid allows the regulation of PEDF expression in transfected pigment epithelial cells. This ability is an important step in the development of a rational cell-based therapeutic approach for the treatment of ocular neovascular diseases.
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