Human breast adenocarcinoma MCF7 cell line (ATCC® HTB22™) and human immortalized keratinocytes HaCaT (CLS # 300493) were used. pLKO.1 lentiviral DNA constructs together with the pΔR8.2 (#12263, Addgene) and pVSV-G (#8454, Addgene) packaging plasmids were transfected into 293FT packaging cells (R70007, ThermoFisher) using TurboFect Transfection Reagent (R0531, Thermo Scientific). Virus-containing supernatants were collected 24 to 48 h after transfection and used to infect the recipient cells in the presence of 8 μg/mL polybrene (SIGMA). Infected cell cultures were selected for 4-5 days in medium containing 1 μg/mL puromicin (SIGMA) for pLKO.1-puro constructs. All experiments were performed 5-8 days after vector-mediated gene transfer.
293ft packaging cell
Manufactured by Thermo Fisher Scientific
Sourced in United States
293FT packaging cells are a specialized cell line used in molecular biology research. They are designed to facilitate the production of recombinant viral particles, such as lentivirus or adenovirus, which can be used for gene delivery or other applications. The 293FT cell line is derived from human embryonic kidney cells and has been engineered to provide high levels of the SV40 large T antigen, which enhances viral packaging efficiency. These cells can be used as a tool to generate high-titer viral stocks for various research purposes.
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Lab products found in correlation
Polybrene, by Merck Group (3 mentions)
Puromicin, by Merck Group (3 mentions)
Pvsv g, by Addgene (3 mentions)
Turbofect transfection reagent, by Thermo Fisher Scientific (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Du145, by American Type Culture Collection (2 mentions)
Penicillin streptomycin, by Corning (2 mentions)
Lncap, by American Type Culture Collection (2 mentions)
Nci h660, by American Type Culture Collection (2 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (2 mentions)
Pten p8, by American Type Culture Collection (2 mentions)
Htb 22, by American Type Culture Collection (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Pmdlg prre, by Addgene (1 mentions)
Pmd2 g, by Addgene (1 mentions)
Prsv rev, by Addgene (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Polybrene, by Nacalai Tesque (1 mentions)
Virapower packaging mix, by Thermo Fisher Scientific (1 mentions)
23 protocols using 293ft packaging cell
1
Lentiviral Transduction of Human Cell Lines
2
Lentiviral Transduction of Mammalian Cells
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Lentiviral particles were produced in human embryonic kidney 293FT packaging cells (Thermo Fisher Scientific, R70007) by cotransfecting with the third-generation lentiviral packaging system, pMDLg/pRRE (Addgene, plasmid #12251), pRSV-Rev (Addgene, plasmid #12253), and pMD2.G (Addgene, plasmid #12259), along with the pLenti6.3/TO/V5-DEST-mTurquoise2, pLenti6.3/TO/V5-DEST-IGFBP2, and pLenti6.3/TO/V5-DEST-IGFBP2-Clover transfer plasmids using Lipofectamine 3000 (Thermo Fisher Scientific) in OptiMEM (Gibco, 31985070), as per the manufacturer’s protocol. Twenty-four hours after transfection, the medium was replaced with TIF growth medium and incubated for a further 24 hours, at which point the medium was collected and filtered through a 0.45-μm syringe filter. MM231s or TIFs were then transduced with the lentiviral particles for 48 hours, in the presence of polybrene (8 μg/ml; Sigma-Aldrich, TR-1003-G), before washing and selecting stable positive cells using puromycin (1 μg/ml).
3
Lentivirus-Mediated Gene Transfer Protocol
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pLKO.1 lentiviral DNA constructs together with the pΔR8.2 (#12263, Addgene) and pVSV-G (#8454, Addgene) packaging plasmids were transfected into 293FT packaging cells (R70007, ThermoFisher) using TurboFect Transfection Reagent (R0531, Thermo Scientific). Virus-containing supernatants were collected 24 to 48 h after transfection and used to infect the recipient cells in the presence of 8 μg/mL polybrene (SIGMA). Infected cell cultures were selected for 4–5 days in medium containing 1 µg/mL puromicin (SIGMA) for pLKO.1-puro constructs. All experiments were performed 4–6 days after vector-mediated gene transfer.
4
Lentiviral Transduction Protocol
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Lentivirus was produced using 293FT packaging cells (Thermo Fisher Scientific) and transduction conducted as described in the Supplemental Material .
5
Prostate Cancer Cell Line Validation and Analysis
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LNCaP and VCaP cells were obtained from ATCC and 293FT packaging cells from Invitrogen (Carlsbad, CA) and cultured per manufacturers' instructions. LNCaP-Abl cells [9 (link)] were a gift from Dr. Helmut Klocker (University of Innsbruck, Innsbruck, Austria) and cultured in RPMI-1640 supplemented with 10% (v/v) charcoal dextran-stripped serum (CDSS), 0.1 μM sodium pyruvate, 1% GlutaMAX, and antibiotics at 37°C in 5% CO2. The identity of all 4 cell types was authenticated once every year at the MDACC Cell Line Authentication Service Core using the STR (Short Tandem Repeat) method. Antibodies used in this study were presented in Supplementary Table S1 . The candidate library inhibitors and MDV3100 (5 month) hit compounds are presented in Supplementary Table S2 . A full list of the library of kinase inhibitors is shown in Supplementary Table S3 and the secondary hit compounds are presented in Table 1 .
6
Lentiviral Transduction with p53 shRNA
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p53 shRNA and GSE22 were previously described43 (link),44 . 293FT packaging cells (Invitrogen) were used to generate lentiviruses.
7
Prostate Cancer Cell Line Culture Protocols
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LNCaP, VCaP, DU145, PC3, NCI-H660, Pten-p8 cells were obtained from ATCC, Cells were cultured in Dulbecco’s modified eagle medium (DMEM; Gibco) or RPMI 1640 medium (Gibco) supplemented with fetal calf serum (FBS, 10%; Gibco), and penicillin-streptomycin (Corning). All cell lines were grown at 37 C, 5% CO2, 95% humidity. 293FT packaging cells were from Invitrogen and cultured according to manufacturers’ instructions. MDA-PCa-2B cells were obtained from Dr. Nora Navone’s laboratory and cultured in BRFF-HPC1 (AthenaES, MD) supplemented with 10% FBS and Gentamycin at 37°C in 5% CO2. RM1 cells were obtained from Dr. Timothy Thompson’s laboratory and cultured in DMEM supplemented with 10% FBS and penicillin-streptomycin at 37°C in 5% CO2.
8
Prostate Cancer Cell Line Culture Protocols
9
Lentiviral Vector Construction with Tnmd Promoter
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To construct lentiviral vector containing Tnmd promoter, the CMV promoter in pLentiLox 3.7 (pLL3.7) which drives GFP gene expression was cut off by NheI and NotI, and about 2kb Tnmd promoter was cloned by PCR and ligated into the plasmid. The primers used for cloning is as following: Forward primer-5’-GGGGATGAACAAGACAGGAAG-3’, Reverse primer-5’-GCAAGTGAGTCGGCTAACAGAT-3’.
Pseudo-lentivirus was produced by transient transfection of 293FT packaging cells (Invitrogen, USA) using the calcium phosphate method. Culture supernatants were harvested at 48 and 72 hours after transfection and lentiviral particles were concentrated using PEG6000 [38 (link)]. For transduction, 1×105 cells were seeded into 6-well plate and incubated with lentivirus and 8 μg/mL polybrene in the incubator for 24h.
Pseudo-lentivirus was produced by transient transfection of 293FT packaging cells (Invitrogen, USA) using the calcium phosphate method. Culture supernatants were harvested at 48 and 72 hours after transfection and lentiviral particles were concentrated using PEG6000 [38 (link)]. For transduction, 1×105 cells were seeded into 6-well plate and incubated with lentivirus and 8 μg/mL polybrene in the incubator for 24h.
10
Lentiviral Transduction Optimization
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Pseudo-lentivirus was produced by transient transfection of 293FT packaging cells (Invitrogen, USA) using the calcium phosphate method. Culture supernatants were harvested at 48 and 72 hours after transfection and lentiviral particles were concentrated using PEG600050 (link). For transduction, 1 × 105 cells were seeded into 6-well plate and incubated with lentivirus and 8 μg/mL polybrene in the incubator for 24 h.
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