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).