Pcmv vsv g envelope

Manufactured by Addgene

Sourced in United States

The PCMV-VSV-G envelope is a plasmid that encodes the vesicular stomatitis virus glycoprotein (VSV-G). This protein is commonly used as a viral envelope for lentiviral and retroviral vector production.

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Lab products found in correlation

Pspax2 packaging, by Addgene (1 mentions) Plko 1, by Thermo Fisher Scientific (1 mentions) Ptet o ngn2 puro, by Addgene (1 mentions) Pcmv dr8, by Addgene (1 mentions) Lenti x concentrator kit, by Takara Bio (1 mentions) Lentivirus qpcr titer kit, by Applied Biological Materials (1 mentions) Control sirna a, by Santa Cruz Biotechnology (1 mentions) Sox 2 sirna, by Santa Cruz Biotechnology (1 mentions) Plko 1 puro lv c, by Thermo Fisher Scientific (1 mentions) Puromycin, by Merck Group (1 mentions) Dh5 alpha cells, by New England Biolabs (1 mentions) Mem neaa, by Thermo Fisher Scientific (1 mentions) Opti mem 1, by Thermo Fisher Scientific (1 mentions) Pspax2 packaging plasmid, by Addgene (1 mentions) Lenti x concentrator, by Takara Bio (1 mentions) Plenti cmv puro dest, by Addgene (1 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions) Fugene 6, by Promega (1 mentions) Sh800s cell sorter, by Sony (1 mentions)

Close spelling variants of this product

PCMV.dR8.2 dvpr lentiviral packaging and pCMV-VSV-G envelope plasmids PCMV-VSV-G envelope plasmid VSV-G envelope PCMV-VSV-G VSV-G

6 protocols using pcmv vsv g envelope

Lentiviral shRNA Knockdown Procedure

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The procedures for shRNA-mediated gene knockdown have been described previously (25 (link)) with some modifications. The shRNA sequences are listed in Supplemental Table S1. To produce lentiviral particles expressing the specific shRNA in a well of a six-well dish, the lentiviral vector pLKO.1 (Life Technologies) with the inserted shRNA-expressing cassette (0.5 μg), together with the pCMV-VSV-G envelope (0.5 μg; Addgene #8454) and the psPAX2 packaging (0.5 μg; Addgene #12260) plasmids, were co-transfected into 293T cells. Sixteen hrs later, cells were changed into fresh medium (DMEM + 20% FBS) and cultured for additional 24 h. The virus-containing medium was then collected, filtered through a 0.45 micron filter and added to the cells to be infected. Spin-infection was performed by incubation of the virus-containing medium with the cells at 25°C for 1.5 h at 1200 rpm. The infected cell pool was obtained after the selection with puromycin (2 μg/ml) for 5 days for C2C12 cells and 3 days for Hela and 293T cells.

Yu D., Cattoglio C., Xue Y, & Zhou Q. (2019). A complex between DYRK1A and DCAF7 phosphorylates the C-terminal domain of RNA polymerase II to promote myogenesis. Nucleic Acids Research, 47(9), 4462-4475.

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Generation of Inducible Neurodegeneration Models

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Novel doxycycline-inducible, FLAG-tagged, full-length WT Tau, TDP-43, and α-syn lentiviral plasmid constructs were first designed and synthesized (Epoch Life Sciences) using a puromycin resistant plasmid backbone, pTet-O-Ngn2-Puro (Addgene #52,047). Using these 3 WT plasmid constructs, individual constructs containing single pathogenic point mutations were also generated: Α-syn A30P, E46K, H50Q, G51D and A53T; TDP-43 G298S, A315T, A321G, Q331K and M337V; and tau K257T, N279K, P301S and S305N. With these constructs, lentivirus was made using the psPAX2 packing (Addgene #12,260) and pCMV-VSV-G envelope (Addgene #8454) plasmids. To generate stable lines, SH-SY5Y cells already containing the pLenti CMV rtTA3 Blast construct (Addgene #26,429) were infected and stable lines (17 in total) were generated using Puromycin 1 mg/mL selection.

Sampognaro P.J., Arya S., Knudsen G.M., Gunderson E.L., Sandoval-Perez A., Hodul M., Bowles K., Craik C.S., Jacobson M.P, & Kao A.W. (2023). Mutations in α-synuclein, TDP-43 and tau prolong protein half-life through diminished degradation by lysosomal proteases. Molecular Neurodegeneration, 18, 29.

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Lentiviral Vector Production Protocol

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Lentiviruses were produced by co-transfecting HEK293T cells on 10-cm dishes with pLKO.1puro transfer, the pCMV-VSV-G envelope and pCMV-dR8.2 packaging plasmids (gift from Dr. Bob Weinberg, [25 (link)], purchased from Addgene) using the calcium phosphate precipitation method. In summary, plasmid DNAs were mixed in sterile distilled water, then 2.5 M CaCl2 was added (final concentration: 125 mM) and the solution was mixed dropwise with 2× HEPES-buffered solution [HBS] (42 mM HEPES, 15 mM D-glucose, 1.4 mM Na2HPO4, 10 mM KCl, 274 mM NaCl 274 mM, pH 7.1). This mixture was added dropwise to attached cells and the medium was replaced with fresh complete DMEM after 6 h. After 48 h had passed post-transfection, the cell medium was collected and centrifuged for 10 min at 3000 rpm, the supernatant was filtered and the lentiviral vector particles were purified and concentrated with a Lenti-X concentrator kit (Takara). After concentration, the viral particles were resuspended in sterile phosphate-buffered saline and the titer of the samples was measured with a qPCR Lentivirus titer kit from Applied Biological Materials (Vancouver, Canada). The samples were stored at −80 °C until the infection of cells.

Gém J.B., Kovács K.B., Szalai L., Szakadáti G., Porkoláb E., Szalai B., Turu G., Tóth A.D., Szekeres M., Hunyady L, & Balla A. (2021). Characterization of Type 1 Angiotensin II Receptor Activation Induced Dual-Specificity MAPK Phosphatase Gene Expression Changes in Rat Vascular Smooth Muscle Cells. Cells, 10(12), 3538.

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SOX2 Silencing and Overexpression in Cell Lines

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SOX2-silenced A375-M6 cells were obtained by siRNA transfection with Sox-2 siRNA (sc-38408, Santa Cruz Biotechnology, Dallas, Texas, USA) or control siRNA-A (sc-37007, Santa Cruz Biotechnology), according to manufacturer’s instructions. SOX2 silencing in SSM2c cells was obtained by lentiviral transduction. Lentiviruses were produced in HEK-293 T cells. Lentiviral vectors used were pLKO.1-puro (LV-c) (Open Biosystems, Lafayette, CO, USA) and pLKO.1-puro-shSOX2–1 (LV-shSOX2–1) targeting the 3′ untranslated region of SOX2 (targeting sequence 5’-CTGCCGAGAATCCATGTATAT-3′) as previously reported [13 (link)]. SOX2 overexpression in 501-Mel cells was obtained by retroviral transduction. Retroviruses were produced in HEK-293 T cells. Retroviral vectors used were generated by co-transfection of 1 μg pBABE (Addgene, Cambridge, MA, USA, #1764) or pBABE-SOX2 (cloned into the BamHI/SalI restriction sites of pBABE vector using the following primers: SOX2-F 5’-ATGTACAACATGATGGAGACGG-3′ and SOX2-R 5’-TCACATGTGTGAGAGGGGC-3′), 0.9 μg pUMVC packaging plasmid (Addgene, #8449) and 0.1 μg pCMV-VSV-G envelope (Addgene, #8454).

Andreucci E., Pietrobono S., Peppicelli S., Ruzzolini J., Bianchini F., Biagioni A., Stecca B, & Calorini L. (2018). SOX2 as a novel contributor of oxidative metabolism in melanoma cells. Cell Communication and Signaling : CCS, 16, 87.

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Lentiviral knockdown of eIF4E in VAL and OCI-LY1 cells

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The pLKO.1 lentiviral vectors with eIF4E and scrambled shRNA (Sigma Aldrich, St. Louis, MO) were used with pCMV-VSVG envelope (Addgene plasmid 8454,Weinberg lab) and psPAX2 packing plasmids (Addgene plasmid 12260, Trono lab) to generate infectious lentiviral particles. VAL and OCI-LY1 cell lines were infected with high titer lentivirus and selected with 2 µg/ml puromycin (Sigma Aldrich). The knockdown of eIF4E was confirmed by western blotting.

Mallya S., Fitch B.A., Lee J.S., So L., Janes M.R, & Fruman D.A. (2014). Resistance to mTOR Kinase Inhibitors in Lymphoma Cells Lacking 4EBP1. PLoS ONE, 9(2), e88865.

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Generating Lentiviral Particles for Transduction

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sfGFP plasmid^{51 (link)} gifted by David Thompson from the Church Lab was cloned into a pLenti CMV Puro DEST (Addgene, 17452) vector using LR Clonase II and transformed into DH5alpha cells (NEB, C2987H). Purified pLenti-sfGFP plasmid DNA (37.5 ug) was diluted into 1 ml Opti-MEM I (ThermoFisher, 319850672) with lentivirus pCMV-VSV-G envelope (Addgene, 8454) and psPAX2 packaging plasmid (Addgene, 12260) in a 3:3:1 mass ratio. FuGene6 (56.25 μl; Promega, E2691) was added to the mixture and incubated for 20 min at r.t. The transfection mix was then diluted in 15 ml antibiotic-free media consisting of Dulbecco’s modified Eagle medium (DMEM), high glucose (Gibco, 11965118), 10% fetal bovine serum, 0.1 mM minimal essential media non-essential amino acids (MEM-NEAA) (Gibco, 11140050), 6 mM l-glutamine, and 1 mM sodium pyruvate, and added to a T75 flask that was plated 1 d before transfection with 5.5 million human embryonic kidney (HEK) 293T cells. After 3 d, media with packaged lentivirus were collected and concentrated 50× using Lenti-X concentrator (Takara Bio, 631231), aliquoted and frozen at −80 °C. Single-cell clones of PGP1-SV NGN1 were subjected to transduction by packaged lentivirus for 1 d. Cells were grown until confluent, flow-sorted in a Sony SH800S cell sorter for the top 10% of sfGFP+ cells, expanded and frozen.

Skylar-Scott M.A., Huang J.Y., Lu A., Ng A.H., Duenki T., Liu S., Nam L.L., Damaraju S., Church G.M, & Lewis J.A. (2022). Orthogonally induced differentiation of stem cells for the programmatic patterning of vascularized organoids and bioprinted tissues. Nature biomedical engineering, 6(4), 449-462.

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