The pX1665 plasmid, containing the anti-HER2 scFv and the HIV protein gp41, was synthesized by Synbio Tech. (South Brunswick, NJ, USA). The pMDLg/pRRE plasmid (Addgene, Watertown, MA, USA) codes for the Gag, Pol and Rev response element (RRE). The pRSV-REV plasmid (Addgene, USA) encodes the Rev protein. The plasmids were replicated in NEB® Stable Competent Escherichia coli, and the cells were transformed by electroporation, in accordance with the protocol described by Lessard [18 (link)].
Prsv rev plasmid
Manufactured by Addgene
Sourced in United States, Switzerland
The PRSV-Rev plasmid is a DNA vector that contains the genetic sequence for the Rev protein from the Primate Sarcoma Virus (PRSV). This plasmid can be used for research purposes to study the function and properties of the Rev protein.
Automatically generated - may contain errors
Lab products found in correlation
Pmdlg prre plasmid, by Addgene (5 mentions)
Pmd2 g plasmid, by Addgene (3 mentions)
Pgl3 plane promoter, by Sangon (2 mentions)
Pgl3 plane promoter e2f1 br, by Sangon (2 mentions)
Pmd2 g envelope plasmid, by Addgene (2 mentions)
Pspax2 packaging plasmid, by Addgene (2 mentions)
Pmd2 g plasmid 12259, by Addgene (1 mentions)
Xfect transfection reagent, by Takara Bio (1 mentions)
Pvdf membrane, by Avantor (1 mentions)
7 protocols using prsv rev plasmid
1
Plasmid Synthesis and Transformation
2
Plasmid Construction and Sources
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The FH1-tUTG plasmid was a kind gift from A/Professor M. J. Herold (Walter and Eliza Hall Institute of Medical Research, Australia). The pcDNA3.1(+) and pEGFP-C1 plasmids were kind gifts from Professor Mian Wu (Translational Research Institute, Henan Provincial People's Hospital and People's Hospital of Zhengzhou University, Zhengzhou, P.R. China) and A/Professor Yongyan Wu (Department of Otolaryngology, Shanxi Key Laboratory of Otorhinolaryngology Head and Neck Cancer, the First Affiliated Hospital, Shanxi Medical University, Taiyuan, P.R. China), respectively. The pMDLg/pRRE plasmid (#12251, RRID: Addgene_12251), pMD2.g plasmid (#12259, RRID: Addgene_12259), and pRSV-Rev plasmid (#12253, RRID: Addgene_12253) were purchased from Addgene. The pEGFP-C1-eEF1α1-FL, pEGFP-C1-eEF1α1-D1, pEGFP-C1-eEF1α1-D2, pEGFP-C1-eEF1α1-D3, pEGFP-C1-eEF1α1-D1-Δ14-21, pEGFP-C1-eEF1α1-D1-Δ91-95, pEGFP-C1-eEF1α1-D1-Δ153-156, pcDNA3.1-MILIP, pcDNA3.1-MILIP-ΔE1, pcDNA3.1-MILIP-ΔE2, pcDNA3.1-MILIP-Δ-990/-1895, pcDNA3.1-MILIP-Δ-1488/-1895, pcDNA3.1-MILIP-R-FL, pcDNA3.1-MILIP-R-ΔE1, pcDNA3.1-MILIP-R-Δ-990/-1488, pcDNA3.1-MILIP-R-ΔDFOs, pcDNA3.1-MILIP-R1, and pcDNA3.1-MILIP-R2 plasmids were purchased from Sangon Biotech.
3
Plasmid Acquisition and Usage Protocol
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The FH1-tUTG plasmid was a kind gift from A/Professor M. J. Herold (Walter and Eliza Hall Institute of Medical Research, Australia). The pcDNA3.1(+), pGL4.73[hRluc/SV40] and pSin-3 × Flag-E2F1 plasmids were kind gifts from Professor Mian Wu (Translational Research Institute, Henan Provincial People’s Hospital and People’s Hospital of Zhengzhou University, Zhengzhou, China). The pEGFP-C1 plasmid was a kind gift from A/Professor Yongyan Wu (Department of Otolaryngology, Shanxi Key Laboratory of Otorhinolaryngology Head and Neck Cancer, the first affiliated hospital, Shanxi Medical University, Taiyuan, China). The pMDLg/pRRE plasmid (#12251), pMD2.g plasmid (#12259) and pRSV-Rev plasmid (#12253) were purchased from Addgene. The pGL3-PLANE-promoter and the pGL3-PLANE-promoter-∆E2F1-BR were purchased from Sangon Biotech (Shanghai, China). Other plasmids used in this study were generated by inserting the PCR products to the pcDNA3.1(+) or pEGFP-C1 vectors. Primers used in the fusion PCR are shown in Supplementary Table 10 .
4
Lentiviral Vector Generation Protocol
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LVs were generated using the transient transfection protocol,64 as described previously. Briefly, 15 μg of vector plasmid, 10 μg of psPAX2 packaging plasmid (Addgene 12260), 5 μg of pMD2.G envelope plasmid (Addgene 12259), and 2.5 μg of pRSV-Rev plasmid (Addgene 12253) were transfected into 293T cells. Vector particles were collected from filtered conditioned medium at 72 h post transfection. The particles were purified using the sucrose-gradient method and concentrated >250-fold by ultracentrifugation (2 h at 20,000 rpm). Vector and viral stocks were aliquoted and stored at −80°C.
5
Lentiviral Particle Production Protocol
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To produce lentiviral particles, 5.5 × 106 293FT cells were plated in a 10 cm dish a day before the transfection. On the following day, cells were transfected using Xfect transfection reagent (Takara Bio, 631318) with 20 μg of lentiCRISPR–Cas9 plasmid, 3.6 μg of pMD2.G plasmid (Addgene, 12259), 3.6 μg of pRSV-Rev plasmid (Addgene, 12253) and 3.6 μg of pMDLg/pRRE plasmid (Addgene, 12251). The medium was changed with complete culture medium 6 h after transfection. The viral supernatant was collected 48 h post-transfection, passed through a 0.45-μm syringe filter (PVDF membrane; VWR, 89414-902), pooled, and used either fresh or snap frozen.
6
Lentiviral Vector Generation Protocol
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Lentiviral vectors were generated using the transient transfection protocol, as described previously.33 (link) Briefly, 15 μg vector plasmid, 10 μg psPAX2 packaging plasmid (Addgene #12260 generated in Dr. Didier Trono’s lab, EPFL, Switzerland), 5 μg pMD2.G envelope plasmid (Addgene #12259, generated in Dr. Trono’s lab), and 2.5 μg pRSV-Rev plasmid (Addgene #12253, generated in Dr. Trono’s lab) were introduced into 293T cells by transfection. To generate IDLVs, a pBK43 (integrase-deficient) packaging cassette was used. Vector particles were collected from filtered conditioned medium at 72 hr post transfection. When necessary, the particles were purified using the sucrose-gradient method33 (link) and concentrated >100-fold by ultracentrifugation (2 hr at 22,000 rpm). Vector and viral stocks were aliquoted and stored at −80°C.
7
Plasmid and Primer Acquisition Protocol
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The FH1-tUTG plasmid was a kind gift from A/Professor M. J. Herold (Walter and Eliza Hall Institute of Medical Research, Australia). The pcDNA3.1(+), pGL4.73[hRluc/SV40] and pSin-3×Flag-E2F1 plasmids were kind gifts from Professor Mian Wu (Translational Research Institute, Henan Provincial People's Hospital and People's Hospital of Zhengzhou University, Zhengzhou, China). The pEGFP-C1 plasmid was a kind gift from A/Professor Yongyan Wu (Department of Otolaryngology, Shanxi Key Laboratory of Otorhinolaryngology Head and Neck Cancer, the rst a liated hospital, Shanxi Medical University, Taiyuan, China). The pMDLg/pRRE plasmid (#12251), pMD2.g plasmid (#12259) and pRSV-Rev plasmid (#12253) were purchased from Addgene. The pGL3-PLANE-promoter and the pGL3-PLANE-promoter-∆E2F1-BR were purchased from Sangon Biotech (Shanghai, China). Other plasmids used in this study were generated by inserting the PCR products to the pcDNA3.1(+) or pEGFP-C1 vectors. Primers used in the fusion PCR are shown in Supplementary Table 9.
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