SARS CoV-2 pseudotyped lentiviruses were produced by transfecting the 293T cells with the pLenti-Puro vectors (Addgene) expressing Luciferase or β-Galactosidase, with pcDNa3.1 vector expressing SARS-CoV-2 spike (BEI repository) and the helper plasmid pSPAX2 (Addgene). The VSV-G and empty lentiviruses were produced by replacing pCDNA3.1-Spike with pcDNA3.1-VSV-G or pCDNA3.1 empty vector, respectively (Addgene). The transfections were carried out using the Polyethylenimine (PEI) method with the ratio at PEI:pLenti:pcNDA3.1-Spike:pSPAX2 = 14:2:2:1 or PEI:pLenti:pVSV-G/pcNDA3.1:pSPAX2 = 10:1:0.5:3. The virus-containing medium was harvested 72 hours after transfection and subsequently pre-cleaned by centrifugation (3,000 g) and a 0.45 μm filtration (Millipore). The virus-containing medium was concentrated by using a LentiX solution (TakaraBio) a 10:1 v/v ratio and centrifuged at the indicated RCF at 4 °C. After centrifugation, the supernatant was carefully removed and the tube was drained on the tissue paper for 3 minutes. Dulbecco’s modified Eagles medium containing 4.5 g/l Glucose (DMEM) was added to the semi-dried tube for re-suspension and then stored at −80 °C.
Plenti puro vector
Manufactured by Addgene
Sourced in United States
The PLenti-puro vector is a lentiviral expression vector designed for the stable integration and expression of genes of interest in target cells. The vector contains a puromycin resistance gene for selection of transduced cells. This vector is suitable for use in lentiviral-based gene delivery and expression applications.
Automatically generated - may contain errors
Lab products found in correlation
Heracell 150i, by Thermo Fisher Scientific (3 mentions)
Pcdna3.1 spike, by Addgene (2 mentions)
Pcdna3.1 vsv g, by Addgene (2 mentions)
Pcdna3.1 empty, by Addgene (2 mentions)
Lenti x solution, by Takara Bio (2 mentions)
Pspax2, by Addgene (2 mentions)
Entrez gene id 967, by GenScript (2 mentions)
Millex hv filter, by Merck Group (2 mentions)
0.45 μm filtration, by Merck Group (1 mentions)
Lenti x gostix plus, by Takara Bio (1 mentions)
Pegfp c1 vector, by Addgene (1 mentions)
Plko 1 trc vector, by Addgene (1 mentions)
Sirna sequence design software, by Takara Bio (1 mentions)
Pgl3.0 basic vector, by Promega (1 mentions)
Pegfp c1 er alpha, by Addgene (1 mentions)
Pbabe puro vector, by Addgene (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Plko 1 puro, by Addgene (1 mentions)
Plenti puro, by Addgene (1 mentions)
Xbai, by Takara Bio (1 mentions)
13 protocols using plenti puro vector
1
Production of SARS-CoV-2 Pseudotyped Lentiviruses
2
Production of SARS-CoV-2 Pseudotyped Lentiviruses
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SARS CoV-2 pseudotyped lentiviruses were produced by transfecting the 293T cells with the pLenti-Puro vectors (Addgene) expressing Luciferase or β-Galactosidase, with pcDNa3.1 vector expressing SARS-CoV-2 spike (BEI repository) and the helper plasmid pSPAX2 (Addgene). The VSV-G and empty lentiviruses were produced by replacing pCDNA3.1-Spike with pcDNA3.1-VSV-G or pCDNA3.1 empty vector, respectively (Addgene). The transfections were carried out using the polyethylenimine (PEI) method with the ratio at PEI:pLenti:pcNDA3.1-Spike:pSPAX2 = 14:2:2:1 or PEI:pLenti:pVSV-G/pcNDA3.1:pSPAX2 = 10:1:0.5:3. The virus-containing medium was harvested 72 h after transfection and subsequently pre-cleaned by centrifugation (3,000g at 4°C) and a 0.45 μm filtration (Millipore). The virus-containing medium was concentrated by using a LentiX solution (TakaraBio) a 10:1 v/v ratio and centrifuged at the indicated RCF at 4°C for 45 min. After centrifugation, the supernatant was carefully removed, and the tube was drained on the tissue paper for 3 min. Dulbecco’s modified Eagle’s medium containing 4.5 g/L glucose (DMEM) was added to the semi-dried tube for re-suspension and then stored at −80°C.
3
Human TMEM97 ORF Cloning and Expression
4
Stable cell line expressing tagged TRIM11
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Human TRIM11 cDNA was constructed into a pSin-puro vector to generate a stable cell line. An S–Flag–SBP (SFB) tag was ligated to the TRIM11 N-terminus to generate the human TRIM11 cDNA construct in the plenti-puro vector (Addgene, #39481). An HA tag was inserted into the N-terminal pcDNA3.1 (+) vector to establish the human p53 cDNA construct.
5
Luciferase Assay for Autophagy Regulation
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For the luciferase assay, the ATG5 and ATG7 promoter regions (−1510 to −7 and −1538 to -26, respectively) were amplified from genomic DNA using the primer pairs shown in S1 Table and inserted into the pGL3.0-basic vector (Promega, Madison, USA). pOTB7-VCP and pT7T3Pac-MAP1LC3B (Korea Unigene Clone) were subcloned into the pCMV-Flag vector and pEGFP-C1 vector, respectively, and Flag-VCP was subcloned into the plenti-puro vector (Addgene) using primer pairs mentioned in S1 Table . Short hairpin RNAs (shRNAs) against KDM3B and VCP were designed using siRNA sequence design software (Clontech). Double-stranded oligonucleotides for shRNA plasmid construction were produced using primers at the 5′ and 3′ ends (S1 Table ). These oligonucleotides were inserted into the AgeI/EcoRI site of the pLKO.1 TRC vector (Addgene).
6
Constructing Estrogen Receptor Dual-Domain Biosensor
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ERDDB was constructed by amplifying each domain using polymerase chain reaction (PCR), digestion, and ligation, according to the sequence shown in Fig. 2 E. CyOFP1 and HaloTag were amplified from pKK-TEV-CyOFP1 (Addgene #105799) and pHAGE-EFS-MCP-HALOnls (Addgene #121937), respectively, and digested using BamHI and XhoI. The EV linker and P2A peptide sequences were amplified from pGEMT-TPE2A-Mef2c-Tdtomato-Gata4-Tbx5 (Addgene #111818) and pCAGGS-6011nes (Addgene #108652), respectively, and then digested with BspEI/MluI for the α/α and α/β ERDDB or BspEI/NotI for the β/β ERDDB. Nano-luciferase was amplified from pcDNA3.1-CAG-NanoLuc-VChR1-EYFP (Addgene #114112) and digested with NotI/XbaI. ERα and ERβ were amplified from pEGFP-C1-ER alpha (Addgene #28230) and pEGFP-C1-ER beta (Addgene #28237), respectively. For the α/α and α/β ERDDBs, the amplified construct was digested with XhoI/BspEI or XbaI/PspOMI, respectively, and for β/β ERDDB, with XhoI/BspEI or MluI/NotI. For lentiviral production, all ERDDBs were digested with BamHI/PspOMI and ligated into the pLenti-puro vector (Addgene #39481).
7
Lentiviral and Retroviral Vector Construction
8
Lentiviral Vector for tdTomato-CD63 Expression
9
Loss- and Gain-of-Function Experiments in MOVAS Cells
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For loss-of-function experiments using MOVAS cells, mouse short hairpin RNAs (shRNAs) designed with web tools (https://portals.broadinstitute.org/gpp/public/ ) were expressed by lentivirus. Their sequences are listed in Table S2 . The shRNA-expressing constructs were generated using the vector pLKO.1 puro (cat. 8453; Addgene). For gain-of-function experiments, stable cell lines were generated using lentiviral vectors. First, mouse and rat mRNAs were extracted from MOVAS cells and rat primary aortic SMCs, respectively, using TRIzol reagents (cat. 15596018; Thermo Fisher Scientific). Second, reverse transcription was performed with the extracted mRNA, and a full-length cDNA was cloned. Coding sequences were Mouse KLF15 (GenBank: NM_023184.4), rat KLF15 (GenBank: NM_053536.2), mouse LGALS3 (GenBank: NM_001145953.1), and rat LGALS3 (GenBank: NM_031832.1). Mouse SREBP1 (GenBank: XM_006532716.3) cDNA was cloned from the pLKO-puro FLAG-SREBP1 vector (cat. 32017; Addgene). KLF15 and SREBP1 cDNAs were each subcloned into the pLenti-puro vector (cat. 39481; Addgene) with a FLAG tag at the N terminus. Mouse and rat LGALS3 cDNAs were also subcloned into this vector but with a V5 tag at the C terminus. The primers used for cloning are listed in Table S3 .
10
Lentiviral Plasmid-Based Gene Transduction
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All third-generation lentiviral plasmids were obtained from Addgene (Watertown, MA, USA). The envelope (pMD2.G; Addgene plasmid #12259; http://n2t.net/addgene:12259; RRID: Addgene_12259) and packaging plasmids (pRSV-Rev; Addgene plasmid #12253; http://n2t.net/addgene:12253; RRID: Addgene_12253 and pMDLg/pRRE; Addgene plasmid # 12251, http://n2t.net/addgene:12251; RRID: Addgene_12251) were gifts from Dr Tronoa (32 (link)). The transfer plasmid was a gift from Ie-Ming Shih (33 (link)) (pLenti-puro; Addgene plasmid #39481; http://n2t.net/addgene:39481; RRID: Addgene_39481). The pLenti-puro FLAG-tagged FOXL2 was generated by PCR using pcDNA3 FLAG-tagged FOXL2 as the template and the primers FLAG-FOXL2-F and FLAG-FOXL2-R. The PCR products were digested with BamHI and XbaI (Takara Bio) and ligated into the pLenti-puro vector (Addgene). 293T cells were used for lentivirus production by transfection of 12 μg of pMDLg/pRRE and pRSV-Rev, 8 μg of pMD2.G and 16 μg of pLenti-puro empty vector or pLenti-puro-FLAG-FOXL2 for 48 h. Viral supernatant was collected for KGN cell infection in the presence of 10 μg/ml polybrene (H9268, Sigma-Aldrich) (0.5 ml of lentivirus to 5 × 104 cells in 1.5 ml of medium). After 48 h of infection, cells were selected with 2 μg/ml puromycin (P8833, Sigma-Aldrich) for 30 days.
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