Pacad5 9.2 100

cDNA clones encoding mouse Rasd1 and a dnRasd1 were the kind gift of Professor Richard Dorin, University of New Mexico. cDNAs were excised from pcDNA3.1 with restriction enzymes KpnI and XhoI and ligated into compatible restriction sites of adenoviral vector pacAd5.CMV (Cell Biolabs). Adenoviral vector pacAd5.CMV.eGFP was used as a control. The adenoviruses were generated by co-transfection of viral shuttle and backbone (pacAd5 9.2-100) vectors in HEK293T cells by calcium phosphate method in accordance with manufacturer’s guidelines (Cell Biolabs). Adenoviruses were purified by two rounds of CsCl ultracentrifugation and desalted using Slide-A-Lyzer dialysis cassettes (Thermoscientific). The purified viruses were aliquoted and stored at −80 °C. The virus titers were determined in triplicate by standard plaque assay. A multiplicity of infection of 10 was used for cell experiments.

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 .