Answer: TU/ml (transduction units per ml) is a common titration standard for lentivirus. It is a functional titer that represents the number of infectious virus particles, which is lower than the number of physical copies, in a sample. We used GFP expression from infection of our HEK293T cell line expressing ACE2 (catalog number SL221), the receptor for the SARS-CoV-2 virus. After infection, we used fluorescence activated cell sorting (FACS) to count the number of infected cells. Because this titer was determined by FACS, it is a digital determination of titer.

The RFU/ml titer is a functional titer based on firefly luciferase relative fluorescent units/ml, similar to what was shown in a published paper (Ou, X., et al., 2020. Nature Communications doi: 10.1038/s41467-020-15562-9). This is dependent on the type of reagents used for the luciferase assay, however. We measured RFU/ml using our High Sensitivity Firefly Luciferase Kit, but one would expect the RFU titer to be lower using our standard luciferase kit with lower sensitivity. Therefore, it’s more meaningful to compare RFUs between the spike-pseudotyped virus vs. VSV-G virus using the same reagents at the same time.

Answer: The mix contains 3 plasmids. Two of these plasmids expresses rev, and one of the plasmids expresses gag and pol. The 3rd plasmid expresses the envelope protein. This mix is identical to GeneCopoeia's Lenti-Pac™ lentiviral packaging mixe except 1) The VSV-G gene of the Lenti-Pac™ lentivirus packaging kit expressed on the envelope plasmid has been replaced with the Spike protein-expressng gene of SARS-CoV-2; and 2) the mix is formulated in a proprietary ratio.

Answer: The packaging mix is not intended to be used for amplifification and extraction from bacteria. You could do it, but then we could not guarantee that you would get colonies expressing all 3 plasmids. In addition, you would likely not get the correct ratios needed among all 3 to get efficient packaging. We have determined this proprietary ratio for optimal virus packaging. Therefore, if you run out of packaging mix DNA, you would need to order more from GeneCopoeia. However, you can transform bacteria with the transfer plasmid, the plasmid that gets packaged by the mix, because this is a single plasmid.

Answer: No. The GFP control plasmid is included so that you know the kit is functioning properly. You can use the kit to package any 3rd-generation lentiviral expression plasmid, also referred to as the "transfer plasmid". GeneCopoeia provides many clones that express ORFs, shRNAs, miRNAs and miRNA inhibitors, gene promoters, and CRIPSPR sgRNAs in more than 80 lentiviral vector backbones. For more information, visit our lentiviral clone page, or contact inquiry@genecopoeia.com.