Modulating RPE Inflammation via Rap1a

Human primary RPE (hRPE; Lonza, Walkersville, MD) was grown in retinal pigment epithelial cell basal media (RtEBM, Lonza) and cells at passages 4–6 were used in the experiments. To increase active Rap1a in RPE, the cells were transduced with adenoviral constructs expressing GFP-tagged active Rap1a (Ad-63E) or GFP only (Ad-GFP)^{7 (link)}. Forty-eight hours post transduction, cells were incubated with recombinant TNF-α (10 ng/ml, R&D Systems, Minneapolis, MN) or PBS for 24 hours.

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Wang H., Kunz E., Stoddard G.J., Hauswirth W.W, & Hartnett M.E. (2019). Optimal Inhibition of Choroidal Neovascularization by scAAV2 with VMD2 Promoter-driven Active Rap1a in the RPE. Scientific Reports, 9, 15732.

Publication 2019

RtEBM recombinant TNF-α

Adenoviral Cells Epithelial cell Human Rap1a Retinal pigment Tnf α

Corresponding Organization :

Other organizations : University of Utah, University of Florida

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Variable analysis

independent variables

Transduction of RPE cells with adenoviral constructs expressing GFP-tagged active Rap1a (Ad-63E) or GFP only (Ad-GFP)

dependent variables

Active Rap1a levels in RPE cells

control variables

Passage number of RPE cells (passages 4-6)
Incubation time with recombinant TNF-α (24 hours)
Concentration of recombinant TNF-α (10 ng/ml)

controls

Positive control: Cells transduced with adenoviral construct expressing GFP-tagged active Rap1a (Ad-63E)
Negative control: Cells transduced with adenoviral construct expressing GFP only (Ad-GFP)
Control treatment: Cells incubated with PBS instead of recombinant TNF-α

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