0.4 μm pore size

Recombinant Ad-Mertk and Ad-GFP were as previously described [33 (link)]. Recombinant Ad-Tyro3 containing a murine Tyro3 open reading frame (NM_019392) under the control of a CMV promoter was constructed using the ViraPower Adenoviral Expression System (Life Technologies). A full coding Tyro3 cDNA clone (IMAGE: 6834015) with a truncated 3’ untranslated region was ligated between the Sal I to Kpn I sites of pENTR11, subsequent to removal from the vector of the ccdB and CmR genes by EcoR I digestion and religation. The resulting plasmid was recombined with pAD/CMV/V5-DEST. Virions were purified by CsCl gradient centrifugation. The rat kidney fibroblast cell line NRK-49F was obtained from American Type Culture Collection and maintained as recommended by the vendor. NRK-49F cells were plated on 8-well chamber slides and cultured for 24 hours before transduction with recombinant adenoviruses. The next day, cells were used for phagocytosis assays or immunoblotting. Murine primary RPE cells were isolated and cultured as previously described [58 (link)] on 6.5 mm transwell inserts with a 0.4 μm pore size (Corning) for 7 days before transduction with recombinant adenoviruses. Cells were used for phagocytosis assays 3 days after transduction.