2.5 Production of lentiviral particles and generation of stable
cell lines
The full length SARS-CoV-2 spike sequence from SARS-CoV-2 spike plasmid
(a gift from Fang Li, Addgene plasmid #145032) was cloned into pHR-CMV
or pHR-CMV_IRES-EmGFP lentiviral expression plasmid (a gift from A.
Radu Aricescu, Addgene plasmid #113887 and #113888) via EcoRI and AgeI
restriction sites. Lentiviral plasmids encoding ACE2 and TMPRSS2 were
cloned and obtained as described in previous work
(Kim et al., 2021). For lentiviral
packaging, HEK293T cells were co-transfected with the lentiviral
expression plasmids and the packaging plasmid psPAX2 and envelope
plasmid pMD2.G using Lipofectamine 3000 (Thermo Fisher, USA) according
to the manufacturer’s instructions. Supernatants containing the
lentivirus were harvested at 24 h and 48 h post-transfection, combined,
and filtered through 0.45 μm-pore-size filter. To generate stable cell
lines, H1299 or HEK293T cells plated on a 6 well plate with 50-60%
confluence were incubated with 1 ml of the viral supernatants in the
presence of polybrene (Merck, Germany) at a final concentration of 4
μg/ml. After 24 h, the culture supernatants were changed with fresh
medium and further cultured for an additional 2-3 days.