Development and validation of reporter pseudovirus in ACE2-293T
with a floxed reporter.
Given that HEK 293T cells do not express ACE2, we transduced HEK 293T
cells with lentivirus containing human ACE2 cDNA for stable expression
of ACE2 gene and sorted these cells after 7 days of transduction to get
uniform population of ACE2 expressing cells (Figure 2A). For the Gag-CRE
fusion, a cell line was generated by lentiviral integration of a floxed
dsRED/eGFP reporter which were sorted to produce a pure population of
dsRED expressing ACE2-293T (Figure 2B). In all three cases, green
fluorescence is a readout of viral entry. To create a screen that was
sensitive to cells that contained genetic knock outs that blocked viral
entry, we used viral titers that resulted in transduction of more than
90% of the ACE2-293T cells. To compensate for Spike-mNG pseudovirus
having consistently lower titers than the VSVG-mNG we concentrated
Spike-mNG by 50-fold. With this concertation step, all three
pseudoviruses produced similar transduction frequencies with over 90%
of the cells transduced (Figure 2C).