Pcmv vsv g

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PCMV-VSV-G is a plasmid that encodes the vesicular stomatitis virus glycoprotein (VSV-G). It is commonly used as a viral envelope protein for the production of lentiviral and retroviral vectors in cell culture applications.

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9 protocols using pcmv vsv g

Silencing of vGPCR in HEK293T Cells

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vGPCR-specific shRNAs have been cloned in-between AgeI and EcoRI restriction sites of pLKO.1 neo (a kind gift of Sheila Stewart). HEK293T cells have been co-transfected with shvGPCR-pLKO.1 neo, pLP1, pLP2 and pCMV-VSV-G (Invitrogen) using linear PEI (MW 40.000, Polysciences). Virus-containing supernatants were sterile-filtrated (0.45 μm) and concentrated by ultracentrifugation (3 hrs, 50.000 g, 4°C) or used freshly for transduction of vGPCR-TC#1 cells. Transduced cells were selected in medium containing 500 – 1000 μg/ml G418 (Genaxxon) and maintained in medium containing 100 – 250 μg/ml G418.
shRNA oligos (target sequence underlined):

Krause C.J., Popp O., Thirunarayanan N., Dittmar G., Lipp M, & Müller G. (2016). MicroRNA-34a promotes genomic instability by a broad suppression of genome maintenance mechanisms downstream of the oncogene KSHV-vGPCR. Oncotarget, 7(9), 10414-10432.

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Lentiviral and Retroviral Transduction in Breast Cancer Cells

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Plasmids including pCMV-VSV-G (#8454), pCMV-dR8.2 dvpr (#8455), pDONR223-MEKDD (#31202), pDONR223-H-RAS V12 (#31201), pLX302 (#25896), pBabe-puro-BRAF-V600E (#15269), EGFR L858R (#11012), EGFR WT (#11011) and pBabe-puro (#1764) were obtained from Addgene (Cambridge, MA, USA). Lentiviral donor plasmids were removed into the destination vector pLX302 using LR Clonase (Invitrogen, Carisbad, CA, USA). Recombined plasmids were verified by sequencing (Sangon Biotech, Shanghai, China). The infectious lentiviral particles were generated by co-transfecting pLX302 containing gene of interest, pCMV-VSV-G and pCMV-dR8.2 dvpr (at a 5:1:4 ratio) into the 293FT cells (Invitrogen, Carisbad, CA, USA). Retroviral particles were produced by transfection of Phenix293 cells (ATCC, Manassas, VA, USA) with pBabe containing gene of interest. Transfections were carried out using Lipofectamine 2000 according to the manufacture's instruction (Invitrogen, Carisbad, CA, USA). Breast cancer cells were infected with viruses in the presence of 8 μg/mL polybrene (Sigma-Aldrich, St. Louis, MO, USA). After 48 h, stable transfected cells were selected with puromycin (2 μg/mL for T47D cells, 1 μg/mL for MCF-7 cells) until control plates became cleared at 3 days post-treatment.

Xu Y.C., Wang X., Chen Y., Chen S.M., Yang X.Y., Sun Y.M., Geng M.Y., Ding J, & Meng L.H. (2017). Integration of Receptor Tyrosine Kinases Determines Sensitivity to PI3Kα-selective Inhibitors in Breast Cancer. Theranostics, 7(4), 974-986.

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Lentiviral Vector Production Protocol

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Lentivirus was produced by cotransfection of the LentiCRISPR-GFP vector with the packaging plasmids psPAX2 and pCMV-VSV-G (Addgene, 12260 and 8454) into 293T cells using polyethylenimine (PEI, Sigma-Aldrich) transfection. For each flask, 5 μg of lentiviral transfer vector, 3 μg of psPAX2, and 2 μg pCMV-VSV-G were diluted in 500 μL OptiMEM (Invitrogen). PEI of 30 μg was diluted in 500 μL OptiMEM 5 minutes before it was added to the mixture of DNA and PEI. The complete mixture was incubated for 15 minutes before added to cells. After 8 hours, the medium was changed to 15 mL DMEM supplemented with 10% FBS. The virus-containing supernatant was collected at 48 hours and 72 hours after transfection and centrifuged at 1200g at 4°C for 10 minutes to pellet cell debris. The supernatant was filtered through a 0.45 μm filter (MilliporeSigma) and concentrated by ultracentrifuging at 100,000g for 2 hours at 4°C. Subsequently, the lentivirus particles were resuspended in an appropriate volume using RPMI-1640 medium at 4°C overnight. Final viral samples were titrated and stored at −80°C in single-use aliquots.

Hu L., Chen L., Xiao Z., Zheng X., Chen Y., Xian N., Cho C., Luo L., Huang G, & Chen L. (2022). Ablation of T cell–associated PD-1H enhances functionality and promotes adoptive immunotherapy. JCI Insight, 7(2), e148247.

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Generation of CAG-GFP Retroviral Vector

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Two packing cell lines coupled with plasmids pCAG-GFP and pCMV-VSV-G (Addgene, Cambridge, MA, USA) were used to construct the CAG-GFP retroviral vector. First, Platinum-GP cells (a gift from Dr. Guo at Ocean University of China) were co-transfected by pCAG-GFP and pCMV-VSV-G with the facilitation of lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Supernatants collected at 48 and 72 h after transfection were pooled together and filtered through a 0.45 μm syringe filter. The filtered supernatant was centrifuged at 65,000 g for 2 h by using an Optima L-100XP Beckman ultraspeed centrifuge with the rotor SW70 Ti (Beckman Coulter, Brea, CA, USA) at 4°C. The virus-containing pellet was then suspended in 0.1 M phosphate-buffered saline (PBS), transferred into a new tube that was centrifuged for another 2 h at 4°C with the rotor SW41 Ti (Beckman Coulter, Brea, CA, USA). The resulted pellet was re-suspended in 0.1 M PBS and used to infect platinum-E cells (a gift from Dr. Dong of Nanjing Agricultural University) for generating a stable virus-producing cell line. The virus-producing platinum-E cells were frozen at −80°C. Concentrated virus solution was then obtained from the stable virus-producing cells through two-step ultraspeed centrifugation as mentioned above.

Gao F., Song X., Zhu D., Wang X., Hao A., Nadler J.V, & Zhan R.Z. (2015). Dendritic morphology, synaptic transmission, and activity of mature granule cells born following pilocarpine-induced status epilepticus in the rat. Frontiers in Cellular Neuroscience, 9, 384.

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Lentiviral shRNA Knockdown of HRNR

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The three lentivirus plasmids containing human HRNR shRNAs, vector plasmid pLKO.1 puro, packaging plasmid pHR’8.2 deltaR dvpr and pCMV-VSV-G were purchased from Sigma (St. Louis, MO, USA). These plasmids were extracted according to the protocol (GeneJET Plasmid Maxiprep Kit, Thermo SCIENTIFIC). The lentiviral packaging cells, 293 T cells (CRL-3216™), were transfected with the three lentivirus plasmids containing human HRNR shRNAs or vector plasmid pLKO.1 puro and packaging plasmid pHR’8.2deltaR dvpr and pCMV-VSV-G at 70% confluence with the use of Lipofectamine 2000 (Invitrogen, Carlsbad, CA) to produce the lentivirus. Media containing the lentivirus were added to the target cells for 24 h. After 24 h, the original medium was replaced with fresh medium. The cells containing the shRNA constructs were selected in the medium containing puromycin and were cultured for approximately 2 weeks [13 (link)]. The stable cell lines were validated by western blotting.

Fu S.J., Shen S.L., Li S.Q., Hua Y.P., Hu W.J., Guo B, & Peng B.G. (2018). Hornerin promotes tumor progression and is associated with poor prognosis in hepatocellular carcinoma. BMC Cancer, 18, 815.

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Lentiviral Vectors for Gene Delivery

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HIV-1based SIN lentiviral vectors were derived from SINF-MU3-W-S vector backbone. hPV16 E6/E7 was inserted upstream of an encephalomyocarditis virus internal ribosome entry site- (IRES-) yellow fluorescent protein (YFP) gene cassette into SINF-MU3-W-S to generate SINF-MU3-E6E7-IRES-YFPW-S. SINF-MU3-hTERT-IRES-GFPW-S was generated by inserting hTERT cDNA upstream of an IRES-green fluorescent protein (GFP) gene cassette into SINF-MU3-W-S. VSV-G-pseudotyped lentiviral vectors were generated in 150 mm tissue culture dishes by transient cotransfection with (1) VSV-G-expressing construct pCMV-VSV-G (Invitrogen, USA) (66 μg), (2) packaging construct pCMVΔR8.2 (addgene) (48 μg), and (3) lentiviral vector plasmids (pSin hTERT or Psin E6-E7) (66 μg) into subconfluent HEK 293FT cells (Invitrogen) by calcium phosphate precipitation (Clontech, Calphos Mammalian Transfection Kit). The supernatant containing the virus was produced in HEK-293FT, collected, filtered, and used to infect bone marrow cells.

Chiara B., Ilaria C., Antonietta C., Francesca C., Marco M., Lucia A, & Gilda C. (2014). SIRT1 Inhibition Affects Angiogenic Properties of Human MSCs. BioMed Research International, 2014, 783459.

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Overexpressing ChREBPβ in Brown Preadipocytes

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Mouse ChREBPβ was amplified by PCR from pCMV-Flag-ChREBPα, which was a gift from Dr. Donald K. Scott of Icahn School of Medicine at Mount Sinai, and subcloned into the EcoR1 site of the retroviral vector pMSCV-PIG using an In-Fusion Cloning kit (Clontech) to generate a ChREBPβ-expressing plasmid, termed pMSCV-PIG-ChREBPβ, which was verified by DNA sequencing. Retroviral packaging plasmids, pUMVC (#8449) and pCMV-VSV-G (#8454), were obtained from Addgene. To generate pseudotyped virus, pUMVC, pCMV-VSV-G and pMSCV-PIG vectors were co-transfected into sub-confluent HEK293T cells using Lipofectamine 3000 Transfection Reagent (Invitrogen) according to the manufacturer's instructions. The virus stocks were collected at 48 h and 72 h after transfection, filtered through a 0.45 μm filter, and frozen at −80 °C. Brown preadipocytes (20–30% confluent) were infected with retrovirus stocks containing 8 μg/ml polybrene for 12 h, washed and cultured in DMEM.

Song Z., Xiaoli A.M., Li Y., Siqin G., Wu T., Strich R., Pessin J.E, & Yang F. (2022). The conserved Mediator subunit cyclin C (CCNC) is required for brown adipocyte development and lipid accumulation. Molecular Metabolism, 64, 101548.

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Lentiviral Transduction of Fibroblasts to Induce SMC Differentiation

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The human MyoD M3 domain (amino acids 1–62) was fused to full-length cDNAs encoding human MEF2C at the carboxy-terminus to create the fusion gene MEF2C*. pLenti CMV Puro (kind gift from Drs. Campeau & Kaufman, Addgene plasmid # 17448) vector encoding the fusion gene MEF2*, and pReceiver-Lv105 (GeneCopoeia) vector encoding human wild-type genes MYOCD, and GATA6, as well as the “empty vector” virus control, were separately co-transfected with packaging plasmids, psPAX2 (kind gift of Dr. Trono, Addgene plasmid # 12260) and envelope plasmid, pCMV-VSV-G (kind gift of Dr. Weinberg, Addgene plasmid # 8454) into 293T cells using Lipofectamine 2000 (Thermo Fisher Scientific). Human adult dermal fibroblasts (hFBs, Lonza) were seeded at a density of 2.5× 10³ cells/cm² in each well of 48-well, or 24-well plates in human fibroblast medium containing DMEM/F-12, 10% fetal bovine serum (FBS), and Penicillin-Streptomycin (Thermo Fisher Scientific) for the virus transduction. Various combinations of fresh 0.3mL of virus supernatant were added to hFB on day −1 with 10 μg/mL polybrene. The virus supernatant was changed to fibroblast medium on day 0. On day 1, the human fibroblast medium was changed to SMC medium containing DMEM/F-12, 10% KSR, 2 ng/mL Recombinant Human TGF-β1, 10 ng/mL Human PDGF-BB, and 1% Penicillin-Streptomycin, which was replaced every other day thereafter (Figure 1 A).

Hirai H., Yang B., Garcia-Barrio M.T., Rom O., Ma P.X., Zhang J, & Chen Y.E. (2018). Direct reprogramming of fibroblasts into Smooth Muscle-Like Cells with Defined Transcription Factors. Arteriosclerosis, thrombosis, and vascular biology, 38(9), 2191-2197.

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Lentiviral shRNA Production and Transduction

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For virus production, HEK-293 cells (70–80% confluency) were transfected using X-tremeGENE 9 (Roche Diagnostics Ltd., Burgess Hill, UK) according to manufacturer’s manual with 3 μg pCMV-dR 8.91 (packaging vector, Thermo Fisher) + 0.7 μg pCMV-VSV-G (envelope vector, Thermo Fisher) + 3 μg Lentiviral shRNA vectors (three pLKO1-Puro vectors targeting all Fbln2 variants of the mouse under control of the human U6 promotor (Clones: TRCN0000109479, TRCN0000109478 and TRCN0000109476 (Thermo Fisher), or a scr ctrl shRNA pLKO1-Puro control vector (Thermo Fisher)). These were mixed in 100 µl of Opti-MEM (Thermo Fisher), 6 μl X-tremeGENE 9 was added, and the transfection solution applied to the cells. After overnight incubation, the medium was replaced with fresh DMEM/10%FBS medium, and incubated for another 24 hrs, after which the virus-containing medium was collected and filtered using syringe-driven filters (Millipore Ltd., Livingstone, UK). Filtered virus was added to EpH4 cells in a 6-well plate, incubated for 4 hrs at 37 °C in a 5% CO₂ incubator, topped up with DMEM/10% FBS medium + 8 ug/ml polybrene (Sigma) and finally incubated for 24 hrs.
The medium was replaced with fresh medium containing 3 µg/ml puromycin (Sigma) for selection of transduced cells. Cells were routinely passaged with puromycin-medium to maintain the selection.

Ibrahim A.M., Sabet S., El-Ghor A.A., Kamel N., Anis S.E., Morris J.S, & Stein T. (2018). Fibulin-2 is required for basement membrane integrity of mammary epithelium. Scientific Reports, 8, 14139.

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