HEK293T cells were cultured in DMEM medium supplemented with 10% FBS and penicillin/streptomycin solution (all obtained from Invitrogen). Viral vectors were assembled as described in ref. 44 (link). pCMVΔ8.2R viral packaging plasmid and pCMV VSVG plasmid were obtained from Addgene. pUCHR_inLuc genomic plasmid was a kind gift of Dr. D. Mazurov. Viral stocks were concentrated by centrifugation at 30000 g and resuspended in PBS. p24 was assayed using the HIV-1 p24-antigen IFA kit (Vector Best).
Pcmv vsv g plasmid
Manufactured by Addgene
Sourced in United States
The PCMV-VSV-G plasmid is a genetic construct containing the gene encoding the vesicular stomatitis virus glycoprotein (VSV-G). This plasmid is commonly used in research applications that involve the production of lentiviral vectors.
Automatically generated - may contain errors
Lab products found in correlation
Pspax2, by Addgene (4 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (2 mentions)
Pcmvδ8.2r viral packaging plasmid, by Addgene (2 mentions)
Hiv 1 p24 antigen ifa kit, by Vector-Best (2 mentions)
Polyethylenimine (pei), by Polysciences (2 mentions)
Pcmvδ8.2r, by Addgene (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Pspax2 plasmid, by Addgene (2 mentions)
Effectene transfection reagent, by Qiagen (2 mentions)
Pcmv dr8.2 dvpr plasmid, by Addgene (2 mentions)
Amicon ultra 15 ml centrifugal filter, by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Lenti x concentrator, by Takara Bio (1 mentions)
Gag pol expression plasmid, by Addgene (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Lenti xtm 293t cells, by Takara Bio (1 mentions)
Plenti c myc ddk ires puro, by OriGene (1 mentions)
0.45 μm pore size filter, by Corning (1 mentions)
Plvx ires zsgreen1 vector, by Takara Bio (1 mentions)
18 protocols using pcmv vsv g plasmid
1
Lentiviral vector production in HEK293T cells
2
Lentiviral Production and Titration in HEK293T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For lentivirus production, Human Embryonic Kidney 293T (HEK293T) cells were cultured in Dulbecco’s Modified Eagle Medium (Gibco) with 10% FBS and 1% penicillin/streptomycin (Gibco). HEK293Ts were transfected at 70–90% confluency with 10 μg CROP-seq gRNA plasmid, 10 μg Gag-pol expression plasmid (psPax2, gift from Didier Trono, Addgene plasmid #12260), and 1 μg pCMV-VSV-G plasmid (gift from Bob Weinberg, Addgene plasmid #121669) using polyethylenimine (PEI, Polysciences Inc.) at a 3:1 PEI:plasmid ratio. Approximately 6–8 hours after transfection, the medium was aspirated from cells and replaced with Opti-Mem (Gibco). Supernatant containing lentivirus was collected 48 hours after transfection, the medium was replaced, and medium was collected once more after an additional 48 hours. Viral supernatants were filtered through a 0.45 μm PES membrane bottle top filter (Thermo Fisher) and then concentrated with Lenti-X Concentrator (Takara) according to the manufacturer’s instructions. Purified and concentrated lentivirus was used immediately or stored at −80° C. Lentivirus was titered by counting the number of initially transduced cells, adding serial dilutions of lentivirus to primary human T cells, and measuring the percentage of GFP+ cells after three days (only in conditions with <30% GFP+ cells to ensure a majority were single transduction events).
3
Lentiviral Particle Generation and Transduction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To generate lentiviral particles, 8 × 105 293 T cells in a 6-cm dish were cotransfected with 2 μg of HIV-1 packaging plasmid pCMVΔ8.2 R (Addgene number 12263); 0.5 μg of the pCMV-VSV-G plasmid (Addgene number 8454) expressing protein G from vesicular stomatitis virus (VSV-G); and 3 μg of one of the pUCHR transfer vector constructed as described in the previous paragraph. Transfection was performed for 6 h using Lipofectamine 2000 reagent (Thermo Scientific, USA) according to the manufacturer’s instructions, and then the culture medium was replaced. Supernatants containing PVs were harvested and cleared through 0.45-μm-pore-size filters at 54 h posttransfection. The 293T/CD4 or Raji/CD4 cells that we described previously (67 (link)) were first infected with PVs to express CCR5. After immunostaining with respective MAb and positive sorting, cells were transduced to express one of the gp41 peptides. Peptide-expressing cells were then sorted based on mClover fluorescence. Transductions were set up using different doses of PVs. Two to three days postinfection the levels of transduction were quantified by flow cytometry, and samples with less than 30% positive events (low MOI) were selected for sorting.
4
Generating Stable OC2 Cell Lines
5
Generating CEM Cells with LPAP Mutants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The CEM cells with the different LPAP mutants were generated by lentiviral transduction of CEM98 cells. For initiation of HIV-1 infection, HEK293T cells were cotransfected with 0.6 μg of HIV-1 packaging plasmid pCMVΔ8.2R (Addgene), 0.9 μg of pUCHR LPAP IRES GFP transfer vector, and 0.15 μg of pCMV-VSVG plasmid (Addgene) expressing Env G from vesicular stomatitis virus, using Lipofectamine 2000 (Invitrogene). The next day the culture medium was replaced, and cells were grown for another 24 h. Supernatant from the 6-cm dish with transfected HEK293T cells was harvested and clarified through a 0.45 μm pore size filter (Corning). Virus-like particles (VLPs) in the supernatant were concentrated by centrifugation at 100,000 g for 2.5 h and resuspended in 0.5 mL of fresh RPMI culture medium. Freshly prepared VLPs in a volume of 1 mL were added to 1 × 105 CEM cells. Cells were grown for 4 days and sorted by GFP expression.
6
Lentiviral Knockdown and Overexpression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following short hairpin RNA (shRNA) expression sequences were used: DDX52, 5′-GCCAATCCAAATGCAAGCCAT-3′; c-Myc, 5′-CCCAAGGTAGTTATCCTTAAA-3′; and control, 5′-GCTCCGTGAACGGCCACGAGT-3′. These sequences were cloned into the PLKO.1 vector (10879; Addgene, Watertown, MA, USA). The overexpressed DDX52 plasmids were cloned into the pLVX-IRES-ZsGreen1 vector (Takara Bio, Shiga, Japan) via DNA assembly (#E5520; NEB, Rowley, MA, USA).
The psPAX2 plasmid (12260; Addgene) and pCMV-VSVG plasmid (8454; Addgene) in 293 T cells were transfected into HEK293T cells with PEI 25K (23966-1; Polysciences, Warring, PA, USA) following the manufacturer’s instructions. The culture medium was collected after 48 and 72 h, and the supernatant was centrifuged at 1000 rpm for 10 min to obtain the supernatant products containing the lentivirus to transduce into the indicated cells. Puromycin (5 µg/ml) (Sigma–Aldrich, St. Louis, MO, USA) was used to isolate the stable transformants in PC3 and 22RV1 cells.
The psPAX2 plasmid (12260; Addgene) and pCMV-VSVG plasmid (8454; Addgene) in 293 T cells were transfected into HEK293T cells with PEI 25K (23966-1; Polysciences, Warring, PA, USA) following the manufacturer’s instructions. The culture medium was collected after 48 and 72 h, and the supernatant was centrifuged at 1000 rpm for 10 min to obtain the supernatant products containing the lentivirus to transduce into the indicated cells. Puromycin (5 µg/ml) (Sigma–Aldrich, St. Louis, MO, USA) was used to isolate the stable transformants in PC3 and 22RV1 cells.
7
Lentiviral Overexpression of GFP, Trps1, and MGMT
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells overexpressing GFP, Trps1 and MGMT (referred to as Le/GFP, Le/Trps1 and Le/MGMT) were established by lentiviral transduction with each cDNA construct (pLenti‐CMV‐GFP, pLenti‐CMV‐Trps1, or pLenti‐CMV‐ MGMT). In brief, HEK‐293T cells were cotransfected with lentiviral constructs as well as helper vectors pCMV‐VSV‐G plasmid (Addgene) and pCMV‐dR8.2 dvpr plasmid (Addgene). The viral supernatant fraction was collected at 48 h and 72 h after transfection. H446 and H446/CDDP cells were infected with lentiviral particles for 24 h and then selected with puromycin over 1 week. Plasmids were transected using effectene transfection reagent (QIAGEN, Germantown, MD) according to the manufacturer's instructions.
8
Cloning SARS-CoV-2 Spike Protein for Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Original sequence SARS-CoV-2 spike protein (GenBank NC_045512.2) was amplified from puno1-SARS2-S plasmid (InvivoGen, San Diego, CA, USA) and cloned into pSelect gfp plasmid (InvivoGen) BamH1 using Gibson assembly (GA) with the following primers: sprot_fwdgfp: agatcaccggcgtgtcgacgATGTTTGTTTTTCTTGTTTTATTGC and sprot_revgfp: cccatggctgcagagcgctgTTATGTGTAATGTAATTTGACTCCTTTG.
For construct stability, we used NEB stable cells according to manufacturing protocol with small changes (transformation outgrowth performed at room temperature for 2 days. Routine E. coli cultures were grown at 30 °C).
The control pCMV-VSV-G plasmid, lentiviral packaging, and transfer plasmid psPAX2, plenti-CMV-gfp, and pLenti-CMV-luc were obtained from Addgene (Watertown, MA, USA).
For construct stability, we used NEB stable cells according to manufacturing protocol with small changes (transformation outgrowth performed at room temperature for 2 days. Routine E. coli cultures were grown at 30 °C).
The control pCMV-VSV-G plasmid, lentiviral packaging, and transfer plasmid psPAX2, plenti-CMV-gfp, and pLenti-CMV-luc were obtained from Addgene (Watertown, MA, USA).
9
SARS-CoV-2 and VSV-G Pseudovirus Generation and Neutralization Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To generate SARS-CoV-2 or VSV-G pseudovirus particles, suspension cultures of 293T cells were seeded onto a 10 cm dish and transfected with 10 µg of pLVPG (kindly provided by I.V. Zvyagin, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia) plasmid, 8 µg of pCMV-deltaR8.2 plasmid (Addgene, #12263) and 5 µg of pVAX-1-S-glycoprotein (Evrogen, Moscow, Russia) or pCG1-SARS-2-S-delta 18 UK variant or pCG1-SARS-2-S-delta 18 South Africa variant plasmids (kindly provided by Prof. Stefan Pöhlmann) or pCMV-VSV-G plasmid (Addgene, #8454) using Transporter™ 5 transfection reagent. 72 h after transfection the supernatant was harvested, clarified by centrifugation and passing through a 0.45 µm pore size filter, aliquoted and frozen at – 80 °C.
To evaluate the pseudovirus neutralization activity, HDP-2 was incubated with an equal volume of pseudovirus at 37 °C. Then the mixture was transferred to pre-plated 293T/ACE2 cell monolayers in 96-well plates. After incubation for 72 h, the fluorescence of GFP positive cells was determined using a microplate reader (Hidex Sense Beta Plus, Hidex, Turku, Finland).
To evaluate the pseudovirus neutralization activity, HDP-2 was incubated with an equal volume of pseudovirus at 37 °C. Then the mixture was transferred to pre-plated 293T/ACE2 cell monolayers in 96-well plates. After incubation for 72 h, the fluorescence of GFP positive cells was determined using a microplate reader (Hidex Sense Beta Plus, Hidex, Turku, Finland).
10
TTLL6 and CDDP Knockdown/Overexpression Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TTLL6/CDDP knockdown/overexpression stable cell lines were established by lentiviral transduction with indicated constructs. In brief, vectors pCMV-VSV-G plasmid (Addgene), pCMV-dR8.2 dvpr plasmid (Addgene) and pLenti-CMV-CHOP/pLKO.1-TTLL6-shRNA were co-transfected into HEK-293T packaging cells. After 48 hours incubation, the viral supernatant fraction was collected. Then, cells were infected with lentiviral particles for 24 hours following with over one week of puromycin selection. Plasmids were transected using Effectene Transfection Reagent (QIAGEN) according to the manufacturer's protocol.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!