Generation lentiviral SARS-CoV-2 constructs
VIRUS
Plasmids encoding the SARS-CoV-2 open-reading frames (ORFs) and eGFP control are a kind gift of Nevan Krogan (Addgene plasmid #141367–141395). Plasmids were acquired as bacterial LB-agar stabs and used per the provider’s instructions. Briefly, each stab was first seeded into agar LB (Bacto Agar; BD, USA) in 10 cm plates. Then, single colonies were inoculated into flasks containing LB (BD Difco LB Broth, Lennox; BD, USA) and 100 µg/ml penicillin (BI, Israel). Transfection-grade plasmid DNA was isolated from each flask using the ZymoPURE II Plasmid Maxiprep Kit (Zymo Research, USA) according to the manufacturer’s instructions.HEK 293T cells (ATCC, USA) were seeded in 10 cm cell culture plates at a density of 4x10The following day, cells were transfected with a SARS CoV 2 orf-expressing plasmid and the packaging plasmids using the TransIT-LT1 transfection reagent (Mirus Bio, USA) according to the provider’s instructions. Briefly, 6.65 µg SARS CoV 2 lentivector plasmid, 3.3 µg pVSV-G, and 5 µg psPAX2 were mixed in Opti-MEM reduced serum medium (Gibco, USA), with 45 µl of TransIT-LT1, kept at room temperature to complex and then added to each plate. Following 18 hr of incubation, the transfection medium was replaced with 293T medium and virus-rich supernatant was harvested after 48 hr and 96 hr. The supernatant was clarified by centrifugation (500×g, 5 min) and filtration (0.45 µm, Millex-HV, MerckMillipore). All virus stocks were aliquoted and stored at –80 °C.The packaging plasmids (psPAX2 and pVSV-G) are a kind gift from Prof. N. Benvenisti, Stem Cell Unit at The Hebrew University, Jerusalem, Israel.