Editing SARS2 Pseudovirus (section)

== Virus production ==

Pseudotyped lentiviruses can be generated by transfecting 293Ts&nbsp;<br/>1. Seed 293T cells in D10 growth media (see subsection 4.2 for media composition) so that they will be&nbsp;50-70% confluent the next day. For a 6-well plate, this is 5x105 cells per well (2.5x105 cells per mL).<br/>2. At 16-24 hours after seeding, transfect the cells with the plasmids required for lentiviral production.&nbsp;We transfect using BioT (Bioland Scientific) following the manufacturer’s instructions and using the&nbsp;following plasmid mix per well of a 6-well plate (plasmid amounts should be adjusted for larger&nbsp;plates):<br/>• 1 ug of lentiviral backbone–we used either the ZsGreen (NR-52520) or the Luciferase-IRESZsGreen (NR-52516) backbone<br/>• 0.22 ug each of plasmids HDM-Hgpm2 (NR-52517), pRC-CMV-Rev1b (NR-52519), and&nbsp;HDM-tat1b (NR-52518)<br/>• 0.34 ug viral entry protein–either SARS-CoV-2 Spike (NR-52513, NR-52514, or NR-52515),&nbsp;VSV G (positive control), or transfection carrier DNA (Promega E4881) as a negative control.<br/>3. At 18 to 24 hours post-transfection, change the media to fresh, pre-warmed D10.<br/>4. At 60 hours post transfection, collect virus by harvesting the supernatant from each well and filtering&nbsp;it through a 0.45 um filter. Virus can be stored at 4 C for immediate use or frozen at -80 C. The titers of&nbsp;Spike- and VSV G-pseudotyped lentiviruses were found to be unaffected by a freeze-thaw cycle (data<br/>not shown). All titers presented here are from virus that was frozen at -80 C prior to use.