Rtebm

AAV2-hRPE(0.8)-iCre-WPRE and AAV2-CMV-null were obtained from Vector Biolabs. To generate the OptiDicer virus, first Δhel-DICER1 cDNA was cloned into pAAV-MCS (Agilent Technologies). The total packaging genome size to 5.0 kb. The indicated plasmids were transfected into HeLa (ATCC) and primary human retinal pigmented epithelial cells (hRPE) (Lonza), maintained in RtEBM (Lonza) following the manufacturer’s instructions. Nucleofection with Basic Epithelial Cells Nucleofector Kit (Lonza) was used for transient plasmid transfection with program U-023. The transfection efficiency was >80% as determined by pMaxGFP transfection with fluorescence microscopy. let-7–resistant DICER1 and OptiDicer were synthesized by GeneArt Gene Synthesis (Thermo Fisher Scientific). Expression of OptiDicer was driven by CMV promoter and contained an SV40 polyadenylation signal in the 3′ end. Production and purification of AAV2-OptiDicer were accomplished by Vigene Biosciences.

Human primary RPE cells (hRPE; Lonza, Walkersville, MD) were grown in retinal pigment epithelial cell basal media (RtEBM, Lonza) and used from passages 3–5. In some experiments, the cells were pretreated with the nuclear factor kappa B (NF-ҡB) inhibitor, Bay 11–7082 (5 µM), or Wnt/beta catenin inhibitors, JW 67 (20 µM) or XAV939 (1 µM; Tocris Bioscience, Bristol, UK), and then incubated with human recombinant TNF-α (20 ng/ml, R&D Systems) or PBS (1X, 137.93 mM NaCl, 8.06 mM NaPO₄, 2.67 mM KCl, 1.47 mM KH₂PO₄, pH 7.4) for further analyses.