Viral packaging was performed in LentiX293 cells (Clontech 632180) following transfection with the Lenti-HB9-GFP (Addgene ID 37080) construct and lentiviral packaging plasmids psPAX2 (12259) and pCMV delta R8.2 (12263). Viral preparations were concentrated using LentiX concentrator (Clontech 631231) and tittered by qPCR. Patient-derived neuronal cultures were transduced at a MOI of 5 in the presence of 2µg/mL polybrene. Cells expressing GFP were visualized 10 days after transduction by fluorescent microscopy.
Lenti x 293 cells
Manufactured by Takara Bio
Sourced in Japan
Lenti-X 293 cells are a specialized cell line derived from human embryonic kidney 293 cells, designed for the production of high-titer lentiviral particles. These cells are optimized for efficient lentiviral packaging and are suitable for use in various lentiviral-based applications, such as gene delivery, gene silencing, and cell line generation.
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Lab products found in correlation
Pspax2, by Addgene (4 mentions)
Pmd2 g, by Addgene (3 mentions)
Peg it virus precipitation solution, by System Biosciences (2 mentions)
Mscv n flag ha ires puro, by Addgene (2 mentions)
Lenti x concentrator, by Takara Bio (1 mentions)
X tremegene 9, by Roche (1 mentions)
Pmd2g, by Takara Bio (1 mentions)
A549 cell, by American Type Culture Collection (1 mentions)
Human ab serum, by Gemini Bio (1 mentions)
Superfect, by Qiagen (1 mentions)
Pspax, by Addgene (1 mentions)
Dmem hg, by Thermo Fisher Scientific (1 mentions)
Centricon, by Merck Group (1 mentions)
Ultraculture, by Lonza (1 mentions)
Dna polymerase 1, by New England Biolabs (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions)
Sanger sequencing, by Genewiz (1 mentions)
Fugene hd, by Promega (1 mentions)
Moflo xdp instrument, by Beckman Coulter (1 mentions)
17 protocols using lenti x 293 cells
1
Lentiviral Transduction of Patient-Derived Neurons
2
Lentiviral Transduction of UbV Variants
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Cells stably expressing UbV-AA or UbV-CC4 were generated using lentiviruses. First, Gateway technology was used to transfer the UbV genes into pSCRPSY lentiviral vectors containing red fluorescent protein (RFP) and a puromycin resistance gene (C. Rice, The Rockefeller University, NY). Lentivirus was generated by transfecting Lenti-X 293 cells (Clontech) with packaging vectors expressing vesicular stomatitis virus protein G (VSV-G), pPAX2-HIV-gag (Addgene), and pSCRPSY-FLAG-UbV using XtremeGene9 (Roche). After 3 days, supernatants were collected, centrifuged 5 min at 1,000 × g, and filtered through a 0.45-μm-pore-size filter. HEPES and Polybrene were added to reach final concentrations of 20 mM and 4 μg/ml, respectively. Lentiviruses were stored at −80°C until further use. Stable A549 and A549-RIG-I KO cell lines were generated by puromycin selection after transduction.
3
Lentiviral Particle Production Protocol
4
Glioma Cell Culture Conditions
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Cells were grown in Dulbecco’s modified Eagle’s medium containing 10% FBS and supplemented with 50 µg/ml gentamicin, MEM non-essential amino acids, and 50 µM 2-mercaptoethanol and cultured at 37 °C in an environment containing 5% CO2. The cell culture reagents were obtained from Gibco (Thermo Fisher Scientific). The glioma cell line TB101 and its culture conditions have been previously described35 (link). The primary glioma cell line TB107 was established from a primary human glioblastoma specimen as previously described for TB10135 (link). The TB101 and TB107 cell lines were obtained from glioblastoma patients with informed consent, and the study was approved by the local ethics committee. Lenti-X 293 cells were obtained from Clontech Laboratories, Inc. (Mountain View, CA, USA).
5
Generation and Characterization of SARS-CoV-2 Spike and ACE2 Cell Lines
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Lenti-X 293 cells were purchased from Clontech. A549 cells were obtained from the American Type Culture Collection. Raji cells were kindly provided by the Stem Cell Bank, Chinese Academy of Sciences (Shanghai, China). TF-1 cells were kindly provided by the Cell Resource Center, Peking Union Medical College (Beijing, China). Human NK cells and MSCs were kindly provided by Shanghai Longyao Biotechnology (Shanghai, China). Plasmids encoding the SARS-CoV-2 spike protein and ACE2 were obtained from Molecular Cloud (Nanjing, China) and sub-cloned into a pCDH-EF (System Biosciences) lentiviral vector plasmid with a puromycin-resistance marker. To establish SARS-CoV-2 spike- or ACE2-expressing cell lines, Lenti-X 293, A549, or Raji cells were infected with a SARS-CoV-2 spike- or ACE2-expressing lentivirus. After selection with puromycin, the pooled resistant cells were identified by flow cytometry. The cell culture medium was supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin. Lenti-X 293 as well as A549 cells and their derivatives were cultured in complete Dulbecco’s modified Eagle’s medium (DMEM). Raji cells and their derivatives were cultured in complete Roswell Park Memorial Institute (RPMI) medium. Human AB serum was purchased from Gemini Bio-products (West Sacramento, CA, USA).
6
Production of Pseudotyped Lentiviral Vectors
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Pseudotyped lentiviral particles containing HuR or MEF2C were produced by transfection of pTRIPO‐HuR or pTRIPO‐MEF2C and lentiviral packaging plasmid mixture into Lenti‐X 293 cells (Clontech, Mountain View, CA), following the manufacturer's protocol. Briefly, 6×106 Lenti‐X cells were seeded in 10‐cm plates 24 hours before transfection. Then, cells were transfected using SuperFect (Qiagen) with pTRIPO‐HuR or pTRIPO‐MEF2C (vector plasmids), along with the lentiviral packaging helper plasmids psPAX and pMD2.G. Plasmids psPAX2 (plasmid no. 12260; Addgene, Cambridge, MA) and pMD2.G (plasmid no. 12259; Addgene) were gifts of Dr Didier Trono (School of Life Sciences, École Polytechnique Fédérale de Lausanne, Switzerland). Cell culture medium was changed after 24 hours, and then lentivirus containing medium was collected over 24‐hour periods for 2 days. After collection, PEG‐it Virus Precipitation Solution (System Biosciences, Palo Alto, CA) was added, and the mixture was stored at 4°C overnight, followed by centrifugation to concentrate the lentiviral particles. Concentrated lentiviral solution was stored at −80°C.
7
Cell Culture Protocols for Cardiomyocytes, Myoblasts, and Embryonal Carcinoma
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The rat cardiomyocyte cell line H9c2, the mouse myoblast cell line C2C12, and Lenti-X 293 cells (Takara Bio Inc) were maintained in DMEM supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin, and 100 μg/ml streptomycin under 5% CO2 and 95% air. The mouse embryonal carcinoma cell line P19.CL6 was maintained in MEM-α supplemented with 10% FCS.
8
Lentiviral Vector Production and Characterization
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IgK-Leader sequence with TATp was Gibson cloned into the original pMAL-TatP-mCherry-HA-NLS-ZF00-KRAB via SalI and AscI. The transgene was excised from pMAL backbone via SalI (NEB, Ipswich, MA, United States) and PstI (NEB), followed by blunting with DNA Polymerase I (NEB) cloned into our pCCLc-MNDU3-X2-WPRE vector via SmaI (NEB) restriction site. Clones were subsequently Sanger sequenced confirmed (Quintara Biosciences, South San Francisco, CA, United States). Lentivirus was prepared accordingly to previously published protocols (Kalomoiris et al., 2012 (link)). Briefly, lentivirus was generated by complexing 25 μg of transgene vector, 25 μg of delta 8.9, and 5 μg were VSVG with 145 μg of PEI in 3 ml serum-free DMEM-HG (Thermo Fisher Scientific, Waltham, MA, United States, Catalog #11965118) for 20 min prior to application to 2.5 × 106 Lenti-X 293 cells (Takara Bio, Kusatsu, Japan, Catalog #632180). Twenty-four hours following transfection, cells were switched to serum-free Ultraculture (Lonza, Basel, Switzerland, Catalog #BP12-725F) for 48 h. Viral harvest was performed by transferring the supernatant of transfected Lenti-X 293 cells into 50 ml conical tubes, centrifuged at 500 RPM for 5 min to pellet cellular debris, and virus was isolated in a 100 kDa Centricon (MilliporeSigma, Burlington, MA, United States, Catalog #UFC710008).
9
Lentiviral Transduction of Trophoblast Stem Cells
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Lenti-X 293 cells (Takara, 632180) were transfected with pMD2G (Addgene, 12259), psPAX2 (Addgene, 12260), and shRNA carrying plasmids (Sigma SHC202 for non-specific knockdown; TRCN0000075984 and TRCN0000309816 for GLUL knockdown; TRCN0000031905 and TRCN0000295140 for PEPD knockdown; TRCN0000256401 and TRCN0000256403 for PRODH knockdown) via 1:2:2.5 ratio using Lipofectamine 2000 (Invitrogen, 11668019) according to the manufacturer’s manual. After 24 h transfection, medium was replenished with fresh DMEM medium containing 10% FBS. Lentivirus-containing media was filtered (0.45 μm), mixed 1:1 with fresh media containing 10 μg/ml polybrene, and used to infect TSCs maintained at 32oC. To increase knockdown efficiency infections were repeated at least twice per day. TSCs were recovered in DMEM containing 10% FBS for one day and then split into media containing 2.5 ug/ml Puromycin and cultured at 37 °C for 48 h. Cells were trypsinized and plated in co-culture experiments, or collected for molecular analysis as indicated.
10
Lentiviral Transduction of Hif-1α Constructs
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