Protocol for Adenovirus and AAV Production

HEK293LTV and HEK293Ad cells (Cell Biolabs) were cultivated in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Gaithersburg MD) supplemented with 10% Fetal Bovine Serum (Wisent) 1× Penicillin/Streptomycin (Thermofisher, Rockville, MD), 1× Glutamax (Thermofisher). Cells co-transfected with pAAV2/8 (PennVectorCore), pHelper and pAAV-MCSCII or pAAV-MCSGFP (Cell Biolabs) at a molar ratio of 1:1:1 by Calcium Phosphate (Takara, Ann Arbour, MI) according to manufacturer’s directions. Vector purification and titration were performed as previously described^{23 (link),24 (link)}. Ad5 was produced in HEK293Ad cells. Plasmids pacAd5 CMV K-N: CII and pacAd5 9.2–100 (Cell Biolabs) were co-transfected at a 1:1 molar ratio with Calcium Phosphate (Takara) according to manufacturer’s directions. The virus was released by freeze–thaw and subsequently amplified in HEK293Ad cells and purified as previously described²⁵ .