Vsv g plasmid

Lentivirus preparation and B cell transduction were described previously.^{59 (link)} Briefly, 293T cells were cotransfected with psPax2 plasmid (Addgene), VSVG plasmid (Addgene), siControl plasmid, siHSP70 plasmid or siHSF1 plasmid (ABM, Richmond, BC, Canada), lenti-EV (empty vector) or lenti-HSP70 overexpression plasmid (Sinobiological) using Lipofectamine 2000 (Thermo Fisher). Supernatants were collected and filtered. Isolated bone marrow B cells were transduced by lentiviral particles by spin infection.

To rescue recombinant ΔS-VRP(G) virus, a mixture of 293T cells/Huh7.5 cells in suspension (1 × 10⁶ cells of each cell type) were transfected with 4 μg of ΔS-Luc-GFP bacmid and 1 μg of VSV-G plasmid (Addgene; 138479) using polyethyleneimine (PEI) transfection reagent (Polysciences; DNA/PEI ratio, 1:4). After 5 h posttransfection, the transfection mixture-containing medium was replaced with DMEM/2% FBS. At 72 to 96 h posttransfection, supernatants were collected and kept as ΔS-VRP(G) seed stocks. To amplify ΔS-VRP(G), Huh7.5 cells seeded in 150-mm plates (1.5 × 10⁷) were transfected with 40 μg of VSV-G plasmid using PEI reagent. At 5 h posttransfection, the transfection mixture-containing medium was replaced with DMEM/10% FBS. On the following day, 1 mL of ΔS-VRP(G) seed stock was added to VSV-G transfected Huh7.5 cells and incubated for 2 h. After a washing with phosphate-buffered saline (PBS), infected Huh7.5 cells were placed in 25 mL of DMEM/2% FBS and monitored for GFP expression and cytopathic effects (CPE). At 48 to 72 h postinfection, supernatants were collected, clarified of debris, aliquoted, and stored at −80°C. These stocks were used to perform subsequent infection experiments.

mFT cells were plated and grown to 75% confluency in a 6‐well plate. 0.89 µg VSV‐G plasmid (Addgene, 8454), 1.38 µg Delta 8.9 plasmid, and 1.78 µg of a luc/mCherry lentiviral construct were added to 250 µL of Opti‐MEM (Invitrogen, 31 985 070). In a separate vial, 10 µL of Lipofectamine 2000 (Invitrogen, 11 668 019) was added to 250 µL of Opti‐MEM and incubated for 5 min at room temperature (RT). After incubation, the lentiviral mixture was combined with the Lipofectamine solution and incubated at RT for 20 min. This mixture was then added to one of the wells and incubated for 24 h at 37 °C. The following day, the media was replaced with antibiotic‐free DMEM and incubated at 37 °C for another 24 h. On the fourth day, the media was collected and centrifuged at 1000 rpm for 10 min, and the supernatant was stored before refreshing the media in the plate again with antibiotic‐free DMEM. The media in the other 5 wells of the plate was aspirated and replaced with 600 µL of the collected supernatant from the transfected well in addition to 8 µg mL⁻¹ of polybrene and 1.4 mL of antibiotic‐free DMEM. The same process was repeated the following day. After this procedure, the cells were sorted for mCherry expression at the Brigham & Women's Flow Cytometry Core.

HEK-293T cells were grown to 40% confluence in antibiotic-free DMEM (Life Technologies, Grand Island, NY) supplemented with 5% FBS (Sigma, Saint Louis, MO). To generate lentivirus, cells were transfected with the plasmids, psPAX2 (12259; Addgene, Cambridge, MA), a lentiviral plasmid containing genetic elements of interest, and VSVg plasmid (8454; Addgene, Cambridge, MA) in a 3:3:1 ratio using Fugene6 Transfection Reagent (Promega, Madison, WI) and Optimem (Fisher Scientific; Waltham, MA) following the manufacturer’s protocol. After 48 hours, the supernatant was harvested, centrifuged to remove cellular debris (300xg, 5 minutes), and filtered using a 0.45 μm filter.