Lentivirus production and Titering

https://www.nature.com/articles/gt201323.pdf

We used a third generation lentivirus with components split over several plasmids. A plasmid containing an HIV-based, self-inactivating lentiviral vector, pCSCGW (pCSC-SP-PW-GFP,62) expressing eGFP from a cytomegalovirus promoter served as the viral backbone. The remaining packaging plasmids included the following: pMDL, containing the HIV gagpol gene; pMD2.g, containing envelope proteins (Nipah G, Nipah F or VSVG); and pRSV-REV, containing the HIV rev gene.2 HEK293T cells were seeded at 6.2 105 per well (six well plates) or 1.2 107 cells per T175 flask in D10 media. The following day, the cells were transfected by the calcium phosphate method as previously described.63 The final transfection mixture had a DNA concentration of 25 mg ml 1 and CaCl2 concentration of 125 mM. The optimized plasmid molar ratios were 1:0.55:0.38:0.55 (CSCGW:MD2.g:MDL:REV) and 1:1:0.8:0.38:0.55 (CSCGW: NiV-GD33:NiV-FD25:MDL:REV). Cells were re-fed D10 media 16 h posttransfection. Culture media containing the lentivirus was harvested 48 h post-transfection and filtered with a 0.45 mm cellulose acetate syringe filter to remove debris. When concentrating the lentivirus, filtered lentiviralcontaining media was centrifuged at either 3600 g for 16–18 h (TX-400 rotor, Thermo, Asheville, NC, USA) or at 100 000 g for 1 h (SW40Ti rotor, Beckman Colter, Indianapolis, IN, USA). The supernatant was then removed and the pellet resuspended in one-hundredth of the original volume (120– 250 ml). Aliquots of virus were stored at 80 1C. For infectious titer determination, HEK293 cells were plated in six well plates at a concentration of 1 105 cells/well in D10 media. The following day, serial dilutions of lentivirus-containing media with a final concentration of 8 mg ml 1 polybrene was added to the cells in a final volume of 1 ml. Each dilution had two corresponding wells as replicates. In some experiments, virus was preincubated with soluble human ephrin-B2/Fc chimera (R&D Systems, Minneapolis, MN, USA). The cells were re-fed fresh D10 media after 4 h of virus exposure. Two days later, cells were harvested for flow cytometric analysis using a FACScan with Cellquest software (BD Biosciences, San Jose, CA, USA). Forward and side scatter were used to gate single cells (10 000 counts). The FL1 channel was setup to count GFPpositive cells by adjusting the gate, such that it excludes 499.5% of an untransduced cell population. Transducing units per ml (TU ml 1 ) were determined using the dilution yielding 2–25% GFP-positive cells in the following equation: (dilution %GFP positive 100 000)/100. All titers reported in the present study were determined from aliquots that underwent one freeze/thaw cycle.