Lentivirus concentration kit

HEK293T cells were cotransfected with a lentiviral expression vector, the packaging plasmid psPAX2 (psPAX2, RRID:Addgene_12260, Addegene) and the envelope plasmid pMD2.G (pMD2.G, RRID:Addgene_12259, Addegene) using polyethylenimine (PEI) transfection reagent (Polyscience, Cat # 23966-1) following the manufacturer’s instructions. Lentiviral particles were collected 48 h after the medium change and concentrated using the lentivirus concentration kit (Genomeditech, Cat# GM-040801-100).

To obtain HEK-293T cells with stable expression of ACE2 protein, a lentiviral system bearing ACE2 (Genbank ID: BAJ21180.1) gene was constructed. In brief, HEK-293T cells (ATCC) with 70%–80% confluence in a 10 cm dish were co-transfected with 12 μg of plasmid pHIV-puro encoding RRE and ACE2 genes, 8 μg of plasmid psPAX2 encoding gag and pol, and 4 μg of plasmid VSV-G encoding G glycoprotein of vesicular stomatitis virus(VSVG) using Lipofectamine 3000 Reagent (Invitrogen). Twelve hours later, the medium was changed to fresh DMEM (Gibco) supplemented with 10% fetal bovine serum (Gibco) for another 48 h culturing. Medium containing virus particles was harvested and concentrated using a Lentivirus Concentration Kit (Genomeditech). The concentrated virus particles were used to infect HEK-293T cells under selection pressure of 10 μg/mL puromycin (Beyotime Biotechnology). The transfection efficiency was examined by flow cytometry using S1-mFc recombinant protein (Sino Biological) as primary antibody and FITC-AffiniPure Goat Anti-Mouse IgG (Jackson) as secondary antibody. The resulting bulk transfected population was sorted on a BD FACSJazz Cell Sorter (BD) with the BD FACS™ Sortware. Cells with top 1% fluorescence intensity were retained and expanded for subsequent use.

To generate cells stably expressing shRNAs, HEK293T cells were transfected with the indicated lentivirus expression vector and viral packaging constructs. Forty-eight hours after transfection, the viral medium was collected, and concentrated overnight by Lentivirus Concentration Kit (Genomeditech, China). The lentivirus was centrifuged at 4000 × g at 4 °C for 25 min, and the resuspended precipitation was used to infect the target cells. After 24 h of infection, cells were treated with puromycin (MCE, USA) for 4–5 days.