Pcmvr8

Manufactured by Addgene

Sourced in United States, Switzerland

PCMVR8.74 is a plasmid vector designed for the expression of proteins in mammalian cells. It contains a CMV promoter to drive high-level expression and a multiple cloning site for the insertion of genes of interest.

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Lab products found in correlation

Pmd2 g, by Addgene (34 mentions) Pcmv vsv g, by Addgene (13 mentions) Facsaria 3 cell sorter, by BD (5 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (4 mentions) Pmd2 g vsvg, by Addgene (3 mentions) Fugene 6, by Promega (3 mentions) Lenti x cells, by Takara Bio (3 mentions) Vsv g, by Addgene (3 mentions) Gag pol, by Addgene (3 mentions) Pcag eco, by Addgene (3 mentions) Polybrene, by Merck Group (3 mentions) Fugene hd transfection reagent, by Promega (2 mentions) Fugene hd, by Promega (2 mentions) Pcmv vsv g envelope plasmid, by Addgene (2 mentions) Sorvall wx, by Thermo Fisher Scientific (2 mentions) Fugene 6 transfection reagent, by Promega (2 mentions) Lenti x concentrator, by Takara Bio (2 mentions) Lentiviral vectorbackbone, by Addgene (2 mentions) Ab202894, by Proteintech (2 mentions) A8592, by Merck Group (2 mentions)

65 protocols using pcmvr8

Inducible Lentiviral Expression System

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Lentiviruses for LCLs were produced by co-transfecting HEK293T cells with pCMVR8.74 (a gift from Dider Trono and Yasuo Ariumi; #22036, Addgene, Watertown, MA, USA), phCMV-GALV-MTR (a gift from Daniel Hodson; #163612, Addgene), and a third plasmid (CSII-BNRF1-HA, CSII-CMV-MCS-IRES2-Bsd, CSII-IFI27-Flag, pLV-shIFI27-T2A-mCherry, or pLV-shScramble-mCherry). Lentiviruses for Akata(-) cells were produced by co-transfecting HEK293T cells with pCMVR8.74, pCMV-VSV-G (a gift from Bob Weinberg; #8454, Addgene), and pLV-Tet3G or pLV-TRE-BNRF1-HA.
LCLs were infected with the lentiviruses by spinoculation at 1500 × g for 1.5 h in the presence of 5 μg/mL polybrene (VectorBuilder). After incubation for 3 h, LCLs were resuspended in a fresh medium. At 3 dpi, infected LCLs were incubated with 10 μg/mL blasticidin for at least 10 days.
To establish Tet-BNRF1-HA/ Akata(-) cells inducibly expressing BNRF1-HA, Akata(-) cells were infected with a lentivirus carrying the Tet3G cassette in the presence of 5μg/mL polybrene, and the next day, the culture medium was replaced with fresh medium containing 150 μg/mL hygromycin. After 14 days of culture, cells were infected with a lentivirus carrying the TRE-BNRF1-HA cassette as previously described, and maintained in the presence of 10 μg/mL blasticidin and 150 μg/mL hygromycin.

Sagou K., Sato Y., Okuno Y., Watanabe T., Inagaki T., Motooka Y., Toyokuni S., Murata T., Kiyoi H, & Kimura H. (2024). Epstein-Barr virus lytic gene BNRF1 promotes B-cell lymphomagenesis via IFI27 upregulation. PLOS Pathogens, 20(2), e1011954.

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Efficient Lentiviral Transduction in Neurons

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To express plasmids in neurons, constructs were cloned into lentiviral backbones and co‐transfected with pMD2.G (Addgene, #12259) lentiviral envelope and with pCMV∆R8.2 (Addgene, #12263) Lentiviral packaging into HEK cells. One day after transfection, medium was changed for X10 Opti‐MEM(R) (Gibco) supplemented with 100× Pen/Strep (Gibco). Forty hours after transfection, conditioned medium was harvested and spun down at 1,000 g to remove cell debris. Supernatant was transferred to Amicon spin filters (Millipore, 100 kDa cutoff) and spun down at 4,000 g for 30 min. Centrifugation was repeated until all supernatant passed the filter. Concentrated virus was diluted in PBS and filtered with a 0.2 μm filter (VWR). Virus was stored at −80°C until use.

Brouwer M., Farzana F., Koopmans F., Chen N., Brunner J.W., Oldani S., Li K.W., van Weering J.R., Smit A.B., Toonen R.F, & Verhage M. (2019). SALM1 controls synapse development by promoting F‐actin/PIP2‐dependent Neurexin clustering. The EMBO Journal, 38(17), e101289.

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Engineered HER2 Expression in Primary Cells

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An inducible HER2 expression vector was constructed by subcloning the ERBB2 open reading frame from pDONR223-ERBB2 (23888; Addgene, Cambridge, MA, USA) into pINDUCER21 (46948; Addgene) using the Gateway LR Clonase kit (Thermo Fisher Scientific) following the manufacturer’s guidelines. Lentiviral particles were generated by co-transfecting HEK293T cells with the packaging plasmids pMD2.G (12259; Addgene) and pCMVR8.2 (12263; Addgene) and either pLV-GFP (36083; Addgene), pLV-Azurite (36086; Addgene) or pINDUCER21-ERBB2 using FuGENE HD transfection reagent (Promega, Madison, WI, USA) following the manufacturer’s guidelines. Virus-containing supernatant was collected 48 h post-transfection.
When primary myoepithelial and luminal cells were infected with lentiviral particles, particles were treated with 20 mU/ml neuraminidase (Sigma-Aldrich) at 37 °C for 30 minutes prior to their addition to cells. Particles provided with SMARTchoice promoter selection plates (GE Dharmacon, Lafayette, CO, USA) were also treated with 20 mU/ml neuraminidase prior to their application to cells; fluorescence intensity values were acquired using a FLUOstar Omega fluorescent plate reader (BMG Labtech, Cary, NC, USA).

Carter E.P., Gopsill J.A., Gomm J.J., Jones J.L, & Grose R.P. (2017). A 3D in vitro model of the human breast duct: a method to unravel myoepithelial-luminal interactions in the progression of breast cancer. Breast Cancer Research : BCR, 19, 50.

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Lentiviral Knockdown of RELA in Melanoma

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Three short hairpin RNAs (shRNAs) of RELA, namely shRNA-1 (5′-CGGATTGAGGAGAAACGTAAATTCAAGAGATTTACGTTTCTCCTCAATCCGTTTTTTG-3′), shRNA-2 (5′-GCCTTAATAGTAGGGTAAGTTTTCAAGAGAAACTTACCCTACTATTAAGGCTTTTTTG-3′) and shRNA-3 (5′-GGATTCATTACAGCTTAATTCAAGAGATTAAGCTGTAATGAATCCATTTTTTG-3′), and a scrambled control shRNA (5′-CGGTCCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTTAACCTTAGGTTTTTG-3′), were synthesized by Genewiz, Inc., and were then cloned into PLKO.1-EGFP-T2A-Puro (Addgene, Inc.) via AgeI/EcoRI enzyme digestion sites. High titer production of lentivector was achieved by transiently co-transfecting 293T cells with lentiviral packaging plasmids, 4 µg pCMVR8.2, 1 µg pCMV–VSVG (Addgene, Inc.) and 3 µg RELA-shRNA-expressing constructs via Lipofectamine^® 3000 (1 µl Lipofectamine 3000 per µg plasmid; Invitrogen; Thermo Fisher Scientific, Inc.). At 70–90% confluence, A375 melanoma cells were infected with the aforementioned shRNA lentiviruses with the help of Polybrene with a final concentration of 6 µg/ml (Sigma-Aldrich; Merck KGaA), according to the manufacturer's protocol. RNA was harvested 48 h post-infection to determine the knockdown efficiency using RT-qPCR, as aforementioned.

Liu J., Zhong F., Cao L., Zhu R., Qu J., Yang L., Chen T., Hu Y., Wang Y., Yao M., Xiao W., Li C., Li B, & Yuan Y. (2020). 7-dehydrocholesterol suppresses melanoma cell proliferation and invasion via Akt1/NF-κB signaling. Oncology Letters, 20(6), 398.

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Lentiviral Transfection of A549 and H838 Cells

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HEK293T cells were co-transfected with the lentiviral overexpressing the envelope plasmid pMD2.G and the packaging plasmid pCMV∆R8.2 (both Addgene, Watertown, MA, USA) using Fugene HD (Promega, Madison, WI, USA) and reduced medium. After 24 h, the medium was replaced with complete medium, and viral particles were harvested. After an additional 24 h, the viral particles were used to transfect A549 cells with a final concentration of 0.8 mg/mL of polybrene (Merck, TR-1003-G; Darmstadt, Germany) twice after 6 h. 0,02% Puromycin (A1113803, Gibco) was used to select and culture the transduced A549 cells. Lentiviral constructs for miR-SCR lentiviral non-targeting control (VSC11714), and shMIMIC-miR-147b (GSH11926-213625996) were purchased from Horizon Discovery Ltd. (Cambridge, UK). The lentiviral vectors were also used for stable transfection of H838 cells. Here cells were seeded in a 6 well-plate (1 ×10⁵ cells/well). For transfection, 200 µL of a transfection solution containing 1 µg plasmid, 6 µL Fugene HD transfection reagent, and Opti-MEM reduced serum medium per well was prepared. After 24 h, the medium was changed to each cell line’s culture medium, containing 0,02% Puromycin for selection of positive cell clones. Subsequently, the cell number was then increased under selection pressure and the cells were assayed.

Turkowski K., Herzberg F., Günther S., Weigert A., Haselbauer T., Fink L., Brunn D., Grimminger F., Seeger W., Sültmann H., Stiewe T., Pullamsetti S.S, & Savai R. (2024). miR-147b mediated suppression of DUSP8 promotes lung cancer progression. Oncogene, 43(16), 1178-1189.

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Synthesis and Compound Acquisition Protocol

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CCT251236 was synthesized as described [26 (link)]. Compounds were purchased as described: KRIBB11 (Merck Millipore), bortezomib (Cambridge Bioscience), Z-VAD-FMK and puromycin (InvivoGen), pactamycin and tunicamycin (Sigma). Plasmids were purchased or obtained as described: HSF1 shRNA pLKO.1 plasmids (Thermo Fisher; TRCN0000007480, TRCN0000007484), pLKO.1 empty vector, pLKO.1 GFP shRNA, pCMV-R8.72 lentiviral packaging and pCMV-VSV-G envelope plasmid (Addgene; plasmid ID 10878, 30323, 22036 and 8454).

Fok J.H., Hedayat S., Zhang L., Aronson L.I., Mirabella F., Pawlyn C., Bright M.D., Wardell C.P., Keats J.J., De Billy E., Rye C.S., Chessum N.E., Jones K., Morgan G.J., Eccles S.A., Workman P, & Davies F.E. (2018). HSF1: Essential for Myeloma Cell Survival and A Promising Therapeutic Target. Clinical cancer research : an official journal of the American Association for Cancer Research, 24(10), 2395-2407.

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Generating Stable HSF1 Knockdown Cell Lines

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MES-SA and FU-MMT-1 cells were obtained from ATCC (Manassas, VA, USA). MES-SA cells were grown in McCoy's 5a with 10% fetal bovine serum (FBS), and FU-MMT-1 cells were grown in RPMI-1640 medium with 10% FBS. The medium was supplemented with 100 U/ml of penicillin and streptomycin. The following plasmids were used to transfect in 293T cells and product lentivirus: HSF1 shRNA pLKO.1 plasmids (Thermo Fisher; TRCN0000007480, TRCN0000007484), pLKO.1 empty vector, pLKO.1 GFP shRNA, pCMV-R8.72 lentiviral packaging, and pCMV-VSV-G envelope plasmid (Addgene; plasmid ID 10878, 30323, 22036, and 8454). Stable HSF1 knockdown MES-SA and FU-MMT-1 cells were screened by puromycin.

Han S., Liu Q., Yang Z., Ma J., Liu D., Yan C, & Liang D. (2022). Identification of Ferroptosis-Related Gene Prognostic Signature and HSF1 for Reversing Doxorubicin and Gemcitabine Resistance in Uterine Carcinosarcoma. Disease Markers, 2022, 6400227.

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Lentiviral Transduction of Jurkat Cells

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Lentivirus for the transduction of Jurkat cells was prepared as following. HEK293T cells were transfected with the respective constructs cloned into pCDH-EF1-MCS-T2A-copGFP (SBI, Palo Alto, CA) and the packaging plasmids pMD2.G (Cat #11259, Addgene, Cambridge, MA) and pCMVR8.74 (#22036, Addgene, Cambridge, MA) in HEK293T cells. For primary T cell transduction, 293TN cells (SBI, Palo Alto, CA) were transfected with the respective expression plasmids and the pPackH1 Packaging mix (SBI, Palo Alto, CA) using lipofectamine 3000 (ThermoFisher, Waltham, MA). Virus-containing supernatants were harvested and concentrated prior to transduction of cells.

Baeuerle P.A., Ding J., Patel E., Thorausch N., Horton H., Gierut J., Scarfo I., Choudhary R., Kiner O., Krishnamurthy J., Le B., Morath A., Baldeviano G.C., Quinn J., Tavares P., Wei Q., Weiler S., Maus M.V., Getts D., Schamel W.W, & Hofmeister R. (2019). Synthetic TRuC receptors engaging the complete T cell receptor for potent anti-tumor response. Nature Communications, 10, 2087.

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Lentiviral Transduction of Islet Cells

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GFP-tagged lentiviral plasmid (Origene PS100071) or GFP-tagged Srf lentiviral plasmid (Origene MR208120L2) was transfected with pCMV-R8.74 (Addgene 22036) and pMD2.G expression plasmid into HEK293T cells. Transfection was performed using PEIsolution (1 mg/ml) and lentiviral supernatants were collected at 48 hours and 72 hours after transfection. The lentivirus was further concentrated by ultracentrifugation at 4 °C. The titer ranged from 5x10⁸ to 1x10⁹ TU/ml.
Lentiviral transduction was carried out as follows: after isolation, islets were cultured overnight and treated with accutase for 10 min. 5x10³ dispersed cells were seeded per well in a 96 v-bottom plate (Fisher Scientific, 12565481) and transduced with lentivirus at MOI 5-6 in the presence of 0.8 ng/ml polybrene. Single cells were re-aggregated by centrifugation at 365 g for 5 min, and medium was changed after overnight culture.

Zeng C., Mulas F., Sui Y., Guan T., Miller N., Tan Y., Liu F., Jin W., Carrano A.C., Huising M.O., Shirihai O.S., Yeo G.W, & Sander M. (2017). Pseudotemporal ordering of single cells reveals metabolic control of postnatal beta-cell proliferation. Cell metabolism, 25(5), 1160-1175.e11.

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Genetic Manipulation of PAX8 Expression

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PAX8 shRNA was cloned in pLKO.1 plasmid (Millipore Sigma TRC Clone id TRCN0000021278). Lentiviral particles were generated by transfecting HEK293T cells with third-generation lentiviral packaging constructs pCMV-VSV-G (Addgene #8454) and pCMVR8.74 (Addgene #22036). Viral supernatant was used to transduce cells using polybrene followed by puromycin (1 µg/mL) selection to generate single cell clones. For MOE cells expressing PAX8 promoter: cells were transfected with pGL4-PAX8 promoter-luciferase plasmid (Hygromycin) and pLVX RFP Plasmid (Puromycin). Cells were selected with antibiotics Hygromycin (50 µg/mL) and Puromycin (1µg/mL), followed by clonal selection. Clone verification was tested through luciferase assay.

Salvi A., Hardy L.R., Heath K.N., Watry S., Pergande M.R., Cologna S.M, & Burdette J.E. (2022). PAX8 modulates the tumor microenvironment of high grade serous ovarian cancer through changes in the secretome. Neoplasia (New York, N.Y.), 36, 100866.

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