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Lenti x hek293t cells

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Lenti-X HEK293T cells are a cell line derived from human embryonic kidney cells that have been engineered to efficiently produce lentiviral particles. These cells are designed to facilitate the production of high-titer lentiviral vectors for gene delivery applications.

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Lab products found in correlation

Pspax2, by Addgene (5 mentions) Pmd2 g, by Addgene (4 mentions) Glutamax, by Thermo Fisher Scientific (3 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions) Polybrene, by Merck Group (2 mentions) Hek293a, by American Type Culture Collection (2 mentions) Hek293t, by American Type Culture Collection (2 mentions) Fetal bovine serum (fbs), by Merck Group (2 mentions) Sodium pyruvate, by Thermo Fisher Scientific (2 mentions) Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (2 mentions) Penicillin and streptomycin, by Thermo Fisher Scientific (2 mentions) Tet system approved fbs, by Takara Bio (2 mentions) Dmem glutamax, by Thermo Fisher Scientific (2 mentions) Rpmi 1640, by Thermo Fisher Scientific (2 mentions) Lenti x concentrator reagent, by Takara Bio (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Hepg2 cell, by American Type Culture Collection (1 mentions) Lenti x packaging single shot, by Takara Bio (1 mentions) Plvx ires mcherry, by Takara Bio (1 mentions) Lenti x gostix kit, by Takara Bio (1 mentions)

27 protocols using lenti x hek293t cells

1

Establishing cell models for FXR studies

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HepG2 cells (ATCC, Manassas, VA) were cultured in EMEM with 10% fetal bovine serum (FBS) and maintained at 37°C in 5% CO2. Lenti-X™ HEK293T cells (Takara Bio., Mountain View, CA) were cultured in DMEM with 10% FBS and 4 mM glutamine. Huh7 cells stably expressing rat sodium-taurocholate polypeptide (Huh7-Ntcp) were kindly provided by Dr. Simon Hohenester (University of Munich, Munich, Germany) and were grown in minimal essential medium containing 10% FBS, 1% nonessential amino acids, 2 mM-glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B (20 (link)). Cells were grown to 80% confluence and serum-deprived for 24 hours before treatment with TCA, TDCA, TCDCA, 6-ECDCA, GW4064 or vehicle. In guggulsterone studies, cells were pretreated with 50 μM guggulsterone for 1 hour prior to receiving either DMSO or 6-ECDCA. For siRNA transfection studies, HepG2 cells were transfected with 7.5 pmol of FXR siRNA, 9.5 pmol of SHP or scramble siRNA using Lipofectamine® RNAiMax for 48 hours prior to GW4064 treatment according to the manufacturer’s protocol.
Liu X., Guo G.L., Kong B., Hilburn D.B., Hubchak S.C., Park S., LeCuyer B., Hsieh A., Wang L., Fang D, & Green R.M. (2018). Farnesoid X Receptor Signaling Activates the Hepatic X-box Binding Protein 1 Pathway in vitro and in Mice. Hepatology (Baltimore, Md.), 68(1), 304-316.
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2

Lentiviral Transduction of iPSC-Derived Cells

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Patient and healthy control iPSC-derived fibroblast-like cells were lentivirally transduced. IKKi gene was subcloned into a lentiviral vector, pLVX-IRES-mCherry (Takara), and transfected into Lenti-X HEK293T cells (Takara) with Lenti-X Packaging Single Shots (Takara). Two days later, supernatants were collected and passed through a 0.22 μM filter. Lentivirus titer was measured with Lenti-X GoStix kit (Takara).
Lee Y., Wessel A.W., Xu J., Reinke J.G., Lee E., Kim S.M., Hsu A.P., Zilberman-Rudenko J., Cao S., Enos C., Brooks S.R., Deng Z., Lin B., de Jesus A.A., Hupalo D.N., Piotto D.G., Terreri M.T., Dimitriades V.R., Dalgard C.L., Holland S.M., Goldbach-Mansky R., Siegel R.M, & Hanson E.P. (2022). Genetically programmed alternative splicing of NEMO mediates an autoinflammatory disease phenotype. The Journal of Clinical Investigation, 132(6), e128808.
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3

Lentiviral Particle Production in HEK293T Cells

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Lenti-X HEK293T cells (Takara Bio) were seeded at a density of 4.5 × 106 cells per 10 cm dish 18–24 h prior to transfection. The prepared cells were co-transfected using the TransIT®-Lenti transfection reagent (Mirus Bio) with MISSION® Genomics Lentivirus Packaging Mix (Mirus Bio) and lentiviral plasmids containing i53 variants/libraries of interest. The viral supernatant was collected 48 h after transfection, passed through a 0.45 μm filter (Cytiva), flash frozen, and stored until use at -80 °C. Viral titers were measured by FACS in K562 cells and were typically ~0.5–1.5 × 107 TU/mL.
Perez-Bermejo J.A., Efagene O., Matern W.M., Holden J.K., Kabir S., Chew G.M., Andreoletti G., Catton E., Ennis C.L., Garcia A., Gerstenberg T.L., Hill K.A., Jain A., Krassovsky K., Lalisan C.D., Lord D., Quejarro B.J., Sales-Lee J., Shah M., Silva B.J., Skowronski J., Strukov Y.G., Thomas J., Veraz M., Vijay T., Wallace K.A., Yuan Y., Grogan J.L., Wienert B., Lahiri P., Treusch S., Dever D.P., Soros V.B., Partridge J.R, & Seim K.L. (2024). Functional screening in human HSPCs identifies optimized protein-based enhancers of Homology Directed Repair. Nature Communications, 15, 2625.
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4

Culturing Lenti-X HEK293T and NALM6 Cells

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Lenti-X HEK293T cells (Takara Bio, catalog no. 632180) were maintained in high-glucose Dulbecco’s modified Eagle’s medium with GlutaMAX (Fisher Scientific, catalog no. 10566024), supplemented with 10% FCS, 100 U/ml of penicillin/streptomycin (PenStrep; Fisher Scientific, catalog no. 15140122), 1 mM sodium pyruvate (Fisher Scientific, catalog no. 11360070), 1× minimal essential medium (MEM) nonessential amino acids (Fisher Scientific, catalog no. 11140050), and 10 mM HEPES solution (Sigma-Aldrich, catalog no. H0887–100ML). Cells were passaged every 2 days using Tryple Express (Fisher Scientific, catalog no. 12604013) for dissociation and maintained at <60% confluency.
NALM6 cells were engineered to express NY-ESO-1 peptide in an HLA-A0201 background, recognizable with the 1G4 TCR by the Eyquem laboratory at University of California San Francisco (UCSF) and provided for TCR stimulation coculture experiments. For simplicity, these cells are referred to as NALM6. NALM6 cells were cultured in RPMI (Invitrogen, catalog no. 21870092) supplemented with 10% FCS, 100 U/ml PenStrep (Fisher Scientific, catalog no. 15140122), 1 mM sodium pyruvate (Fisher Scientific, catalog no. 11360070), and 1X MEM nonessential amino acids (Fisher Scientific, catalog no. 11140050), 10 mM HEPES solution (Sigma-Aldrich, catalog no. H0887–100ML), and 2 mM L-glutamine (Lonza Bioscience, catalog no. 17–605E).
Schmidt R., Steinhart Z., Layeghi M., Freimer J.W., Bueno R., Nguyen V.Q., Blaeschke F., Ye C.J, & Marson A. (2022). CRISPR activation and interference screens decode stimulation responses in primary human T cells. Science (New York, N.Y.), 375(6580), eabj4008.
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5

Lentiviral Transduction and Cell Sorting

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For virus generation, Lenti-X HEK 293T cells (Takara Bio) were transfected with lentiviral packaging plasmids and transgenic constructs. Virus was harvested and clarified through a 0.45-μm filter after 3 d. Lentiviral titering was optimized to result in <50% transduction to generate cells with MOI < 1, and cells were infected via centrifugation at 150g for 1 h in 8 μg/ml polybrene (EMD Millipore). After 3 d, transduced cells were selected via FACS using an 85–100 μm-diameter nozzle. ATF4 reporter cell lines were generated and sorted following stress induction as previously described (Guo et al, 2020 (link)).
Park J., Desai H., Liboy-Lugo J.M., Gu S., Jowhar Z., Xu A, & Floor S.N. (2024). IGHMBP2 deletion suppresses translation and activates the integrated stress response. Life Science Alliance, 7(8), e202302554.
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6

Authentication of Cell Lines for Research

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SVG-A cells, HEK293A (CRL-1573), HEK293T (ATCC CRL-11268), Lenti-X HEK293T cells (takara, cat# 632180) and African green monkey (CV-1) (ATCC, CCL-70) cells were used. SVG-A cells are a third-generation sub clone of the original SVG cell line (Major et al., 1985 (link)) and express the astrocyte marker GFAP. SVG-A cells and CV-1 were cultured in MEM supplemented with 10% fetal bovine serum and 1% of Amphotericin B, Penicillin, Streptomycin solution. HEK293A and HEK293T were cultured in DMEM supplemented with 10% fetal bovine serum and 1% of Amphotericin B, Penicillin, Streptomycin solution. All cells were grown in an incubator at 37°c with 5% CO2. SVG-A cells and HEK293A cells were authenticated using the ATCC cell line authentication service (STR profile). They have a 55% match with SVG p.12 (human fetal glial cells, CRL-8621) from ATCC and they are of male origin. HEK293A cells have an 81% match with the parental line and are of female origin. We are in the process of authenticating the other cell lines used in the study.
Assetta B., Morris-Love J., Gee G.V., Atkinson A.L., O’Hara B.A., Maginnis M.S., Haley S.A, & Atwood W.J. (2019). Genetic and Functional Dissection of the Role of Individual 5-HT2 Receptors as Entry Receptors for JC Polyomavirus. Cell reports, 27(7), 1960-1966.e6.
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7

SARS-CoV-2 Virion Preparation and Cell Lines

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LentiX-HEK293T cells (Takara Bio #Z2180N) were maintained in DMEM (high glucose) (Sigma-Aldrich, #6429) containing 10% fetal bovine serum (FBS, Sigma-Aldrich #172012), and 1% penicillin-streptomycin (PS) (Sigma-Aldrich, #P4333). HOS cells stably express human ACE2 and TMPRSS2 (HOS-ACE2-TMPRSS2 cells) were prepared as previously described15 (link). VeroE6/TMPRSS2 cells were obtained from the JCRB Cell Bank of NIBIOHN for SARS-CoV-2 virion preparation.
Nagata K., Utsumi D., Asaka M.N., Maeda R., Shirakawa K., Kazuma Y., Nomura R., Horisawa Y., Yanagida Y., Kawai Y., Sato K., Yamaoka Y., Miyakawa K., Ryo A., Yasutomi Y., Imura A, & Takaori-Kondo A. (2022). Intratracheal trimerized nanobody cocktail administration suppresses weight loss and prolongs survival of SARS-CoV-2 infected mice. Communications Medicine, 2, 152.
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8

Generating and Using Lentiviruses

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Lentiviruses were generated and used as previously described.43 (link) In short, Lenti-X HEK293T cells (Takara, Cat# 632180) were grown in 10 cm dishes to 80% confluency and co-transfected with expression plasmids of interest and second-generation packaging plasmids (psPax2 + pMD2.G) using Lipofectamine 2000. Lentiviral particles were collected from the cell culture supernatant at 24, 48, and 72 h after transfection and concentrated using the Lenti-X concentrator (Contech Takara, Cat# 631231) according to the manufacturer’s instructions. Stably transduced cell lines were generated by lentiviral transduction in the presence of 4 μg/mL protamine sulfate at a multiplicity of infection of three (MOI = 3), followed by purification of mCherry-expressing cells via FACS.
Karnik S.S., Ridge K.D., Bhattacharya S, & Khorana H.G. (1993). Palmitoylation of bovine opsin and its cysteine mutants in COS cells.. Proceedings of the National Academy of Sciences of the United States of America, 90(1), 40-44.
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9

Authentication of Cell Lines for Research

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SVG-A cells, HEK293A (CRL-1573), HEK293T (ATCC CRL-11268), Lenti-X HEK293T cells (takara, cat# 632180) and African green monkey (CV-1) (ATCC, CCL-70) cells were used. SVG-A cells are a third-generation sub clone of the original SVG cell line (Major et al., 1985 (link)) and express the astrocyte marker GFAP. SVG-A cells and CV-1 were cultured in MEM supplemented with 10% fetal bovine serum and 1% of Amphotericin B, Penicillin, Streptomycin solution. HEK293A and HEK293T were cultured in DMEM supplemented with 10% fetal bovine serum and 1% of Amphotericin B, Penicillin, Streptomycin solution. All cells were grown in an incubator at 37°c with 5% CO2. SVG-A cells and HEK293A cells were authenticated using the ATCC cell line authentication service (STR profile). They have a 55% match with SVG p.12 (human fetal glial cells, CRL-8621) from ATCC and they are of male origin. HEK293A cells have an 81% match with the parental line and are of female origin. We are in the process of authenticating the other cell lines used in the study.
Assetta B., Morris-Love J., Gee G.V., Atkinson A.L., O’Hara B.A., Maginnis M.S., Haley S.A, & Atwood W.J. (2019). Genetic and Functional Dissection of the Role of Individual 5-HT2 Receptors as Entry Receptors for JC Polyomavirus. Cell reports, 27(7), 1960-1966.e6.
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10

Lentiviral Transduction and FACS

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For virus generation, Lenti-X HEK 293T cells (Takara Bio) were transfected with lentiviral packaging plasmids and transgenic constructs. Virus was harvested and clarified through a 0.45 μm filter after 3 days. Lentiviral titering was optimized to result in <50% transduction to generate cells with MOI <1, and cells were infected via centrifugation at 150 xg for 1 hr in 8 μg/mL polybrene (EMD Millipore). After 3 days, transduced cells were selected via FACS using a 85–100 μm diameter nozzle.
Park J.E., Desai H., Liboy-Lugo J., Gu S., Jowhar Z., Xu A, & Floor S.N. (2023). IGHMBP2 deletion suppresses translation and activates the integrated stress response. bioRxiv.
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