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Lentix 293 t packaging cells

Manufactured by Takara Bio
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The LentiX-293T packaging cells are a stable cell line derived from human embryonic kidney 293 cells, engineered to produce lentiviral particles. The cells contain the necessary viral packaging components required for the production of recombinant lentiviral vectors.

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Lab products found in correlation

Lipofectamine 2000, by Thermo Fisher Scientific (6 mentions) Polybrene, by Merck Group (4 mentions) Lenti x htx packaging system, by Takara Bio (3 mentions) Puromycin, by Takara Bio (3 mentions) Lenti x htx packaging mix, by Takara Bio (2 mentions) Allophycocyanin conjugated anti rat igm, by Jackson ImmunoResearch (1 mentions) Facsaria 2, by BD (1 mentions) Puromycin, by Thermo Fisher Scientific (1 mentions) Plvx puro, by Takara Bio (1 mentions) Polyethylenimine (pei), by Merck Group (1 mentions) Pspax2, by Addgene (1 mentions) Pmd2 g, by Addgene (1 mentions) Dynabeads human t activator cd3 cd28, by Thermo Fisher Scientific (1 mentions) Fugene hd, by Promega (1 mentions) Tryple express, by Thermo Fisher Scientific (1 mentions)

10 protocols using lentix 293 t packaging cells

1

Lentiviral Transduction of HUVEC Cells

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Empty pLIV and EWSR1-FLI1pLIV–expressing lentiviruses were provided by N. Riggi (University Institute of Pathology Lausanne, Switzerland). Lentiviruses were produced in Lenti-X 293T packaging cells (Takara, Cultek) at a low passage number. For each plate, 7 μg of the lentiviral plasmid, 5 μg of the envelope plasmid (VSV-G), and 6 μg of the packaging plasmid (PAX8) were prepared and introduced by calcium phosphate transfection, according to standard protocols. The supernatant containing lentiviruses was collected 48 hours after transfection. The HUVEC cell line was seeded at 3000 cells/cm2 and transduced with 3:1 of the lentiviral supernatant with fresh media containing Polybrene (Sigma-Aldrich) at 6 μg/ml. Cells were selected with fresh growth media containing puromycin (0.3 μg/ml) for 72 hours. A control dish without the transduction media was also selected with puromycin, to control for killing of nontransduced cells.
Sánchez-Molina S., Figuerola-Bou E., Blanco E., Sánchez-Jiménez M., Táboas P., Gómez S., Ballaré C., García-Domínguez D.J., Prada E., Hontecillas-Prieto L., M. Carcaboso Á., Tirado Ó.M., Hernández-Muñoz I., de Álava E., Lavarino C., Di Croce L, & Mora J. (2020). RING1B recruits EWSR1-FLI1 and cooperates in the remodeling of chromatin necessary for Ewing sarcoma tumorigenesis. Science Advances, 6(43), eaba3058.
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2

Lentiviral Production and GATA-4 Overexpression

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For lentivirus production, pMD2G, pCMV deltaR8.74, or pWPXL- GATA-4 promoter-HA tag-GATA-4-T2A-mCherry were transfected into LentiX-293 T packaging cells (TaKaRa) using Lipofectamine 2000 (Thermo Fisher Scientific). After 3 days, the viral supernatant was collected, centrifuged, and filtered through a 0.45-μm filter. M-HL-1 cells were infected with GATA-4 promoter-HA tag-GATA-4-T2A-mCherry, and then, mCherry-positive cells were isolated by FACS.
Wakao S., Oguma Y., Kushida Y., Kuroda Y., Tatsumi K, & Dezawa M. (2022). Phagocytosing differentiated cell-fragments is a novel mechanism for controlling somatic stem cell differentiation within a short time frame. Cellular and Molecular Life Sciences, 79(11), 542.
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3

Lentiviral Transduction of M-Hepa1-6 Cells

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For lentivirus production, pMD2G, pCMV deltaR8.74, or pWPXL-mCherry were transfected into LentiX-293 T packaging cells (TaKaRa) using Lipofectamine 2000 (Thermo Fisher Scientific). After 3 days, the viral supernatant was collected, centrifuged, and filtered through a 0.45-μm filter. M-Hepa1-6 cells were infected with mCherry-lentivirus and then expanded.
Wakao S., Oguma Y., Kushida Y., Kuroda Y., Tatsumi K, & Dezawa M. (2022). Phagocytosing differentiated cell-fragments is a novel mechanism for controlling somatic stem cell differentiation within a short time frame. Cellular and Molecular Life Sciences, 79(11), 542.
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4

Lentiviral Production and Muse-tk Cell Sorting

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For lentivirus production, pMD2G, pCMV-deltaR8.74, and pWPXL-HSVtk-IRES2-EGFP were transfected into LentiX-293T packaging cells (Takara Bio) using Lipofectamine 2000 (Thermo Fisher Scientific). 3 days after transfection, the viral supernatant was collected, centrifuged, and filtered through a 0.45-μm filter. For human Muse-tk cell sorting, HSVtk-IRES2- EGFP-labeled normal human dermal fibroblasts were incubated with rat anti-SSEA-3 immunoglobulin M (IgM) antibody (1:1,000; BioLegend), detected by allophycocyanin-conjugated anti-rat IgM (1:100; Jackson ImmunoResearch) in the antibody diluents and sorted by Special Order Research Products FACSAriaII (Becton Dickinson, http://www.bd.com) as described previously.27 (link) We also collected ∼5% of cells with the lowest SSEA-3 expression as non-Muse-tk cells. Muse-tk cells and non-Muse-tk cells as a control were used in the following studies.
Yamasaki T., Wakao S., Kawaji H., Koizumi S., Sameshima T., Dezawa M, & Namba H. (2017). Genetically Engineered Multilineage-Differentiating Stress-Enduring Cells as Cellular Vehicles against Malignant Gliomas. Molecular Therapy Oncolytics, 6, 45-56.
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5

Generation of VEGF-Expressing SW480 Cells

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A VEGF-expressing SW480 cell line was generated by lentiviral transduction. Human VEGF-A165 (VEGF) was cloned into the lentiviral expression vector pLVX-Puro (Takara Bio, Gothenburg, Sweden). The expression vector was co-transfected with pMD2.G (AddGene, Watertown, MA, USA) and psPAX2 (AddGene) into Lenti-X™ 293T packaging cells (Takara Bio) using PEI (Sigma Aldrich) at a final concentration of 1uM. The produced virus particles were harvested 48 and 72hrs post transfection and filtered through a 0.4um filter. The SW480 cells were transduced with the produced viruses and a stable population of VEGF expressing cells was isolated by selection with puromycin (2ug/ml for 48h, Thermo Fisher Scientific).
Truelsen S.L., Mousavi N., Wei H., Harvey L., Stausholm R., Spillum E., Hagel G., Qvortrup K., Thastrup O., Harling H., Mellor H, & Thastrup J. (2021). The cancer angiogenesis co-culture assay: In vitro quantification of the angiogenic potential of tumoroids. PLoS ONE, 16(7), e0253258.
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6

Lentiviral Transduction of Primary T Cells

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Lenti-X293T packaging cells (Clontech #11131D) were cultured in medium consisting of Dulbecco’s Modified Eagle Medium (DMEM) (Gibco #10569-010), 10% fetal bovine serum (FBS) (University of California, San Francisco [UCSF] Cell Culture Facility), and gentamicin (UCSF Cell Culture Facility). Fresh packaging cells were thawed after cultured cells reached passage 30.
Pantropic VSV-G pseudotyped lentivirus was produced via transfection of Lenti-X 293T cells with a pHR’SIN:CSW transgene expression vector and the viral packaging plasmids pCMVdR8.91 and pMD2.G using Fugene HD (Promega #E2312). Primary T cells were thawed the same day, and after 24 hr in culture, were stimulated with Dynabeads Human T-Activator CD3/CD28 (Thermo Scientific #11131D) at a 1:3 cell:bead ratio. At 48 hr, viral supernatant was harvested, and the primary T cells were exposed to the virus for 24 hr. At day 5 post T cell stimulation, Dynabeads were removed and the T cells expanded until day 12 when they were rested and could be used in assays. T cells were sorted for assays with a FACs ARIA II on day 5 or 6 post T cell stimulation.
Dannenfelser R., Allen G.M., VanderSluis B., Koegel A.K., Levinson S., Stark S.R., Yao V., Tadych A., Troyanskaya O.G, & Lim W.A. (2020). Discriminatory Power of Combinatorial Antigen Recognition in Cancer T Cell Therapies. Cell systems, 11(3), 215-228.e5.
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7

Propagation of Lenti-X 293T Packaging Cells

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Lenti-X 293T packaging cells (Clontech #11131D) were cultured in medium consisting of Dulbecco’s Modified Eagle Medium (DMEM) (Gibco #10569–010) and 10% fetal bovine serum (FBS) (University of California, San Francisco Cell Culture Facility). Lenti-X 293T cells were cultured in T150 or T225 flasks (Corning #430825 and #431082) and passaged upon reaching 80% confluency. To passage, cells were treated with TrypLE express (Gibco #12605010) at 37 C for 5 minutes. Then, 10 mL of media was used to quench the reaction and cells were collected into a 50 mL conical tube and pelleted by centrifugation (400xg for 4 minutes). Cells were cultured until passage 30 whereupon fresh Lenti-X 293 T cells were thawed.
Kim M.S., Bhargava H.K., Shavey G.E., Lim W.A., El-Samad H, & Ng A.H. (2024). Degron-based bioPROTACs for controlling signaling in CAR T cells. bioRxiv.
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8

Generation of VSV-G Pseudotyped Lentiviruses

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The Lenti-X HTX Packaging System (fourth generation; Clontech) was used to generate recombinant, replication-incompetent VSV-G pseudotyped lentiviruses, according to the manufacturer's instructions. Transfection of the expression vector into the Lenti-X 293T packaging cells (Clontech) was performed in a Lenti-X HTX packaging mix. The supernatants of transfected packaging cells were collected 72 h later and filtered through a 45-μm filter before being added to the cells, with 6 mg/mL polybrene (Sigma-Aldrich). The transduction medium was replaced by a culture medium 24 h thereafter, and cells were then cultured for 72 h to allow gene product accumulation in the cells. Cells were then selected using 1 mg/mL puromycin (Takara Bio Company).
Landau S., Newman A., Edri S., Michael I., Ben-Shaul S., Shandalov Y., Ben-Arye T., Kaur P., Zheng M.H, & Levenberg S. (2021). Investigating lymphangiogenesis in vitro and in vivo using engineered human lymphatic vessel networks. Proceedings of the National Academy of Sciences of the United States of America, 118(31), e2101931118.
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9

Lentiviral Transduction of HAMECs

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The Lenti-X HTX Packaging System (Clontech, a fourth generation) was used to generate recombinant, replication-incompetent VSV-G pseudotyped lentiviruses, according to the manufacturer’s instructions. Transfection of the expression vector into the Lenti-X 293 T packaging cells (Clontech) was performed in a Lenti-X HTX Packaging mix. The supernatants of transfected packaging cells were collected 72 h later and filtered through a 45-μm filter before being added to the HAMECs, with 6 mg/ml polybrene (Sigma-Aldrich). The transduction medium was replaced by culture medium 24 h thereafter, and cells were then cultured for 72 h to allow gene product accumulation in the cells. Cells were then selected using 1 mg/ml puromycin (Takara Bio Company).
Landau S., Guo S, & Levenberg S. (2018). Localization of Engineered Vasculature within 3D Tissue Constructs. Frontiers in Bioengineering and Biotechnology, 6, 2.
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10

Lentiviral Vector Production and Cell Transduction

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Production of lentiviruses was performed according to the manufacturer’s instructions. Briefly, recombinant, replication-incompetent, VSV-G pseudotyped lentiviruses were generated using a Lenti-X HTX Packaging System [Clontech, fourth generation (five vectors split packaging technology and inducible promoters)]. The expression vector (pLVX-tdTomato-C1, Clontech) was transfected, in a Lenti-X HTX Packaging Mix, into the Lenti-X 293T packaging cells (Clontech). Following 72 hours, supernatants containing the lentiviral produced by the transfected packaging cells were filtered through a 45-μm filter to remove cellular debris. HAMECs were seeded at a density of 5000 cells/cm2 and allowed to adhere overnight. Cells were transduced with td-Tomato lentivirus particles in the presence of polybrene (1.5 μg/ml; Sigma-Aldrich) per well. Transduction media were replaced after 24 hours, and the cells were incubated for up to 72 hours to allow for gene products to accumulate within the target cells. Selection for stably expressing cells was performed using puromycin (3 μg/ml; Takara Bio Company).
Graney P.L., Ben-Shaul S., Landau S., Bajpai A., Singh B., Eager J., Cohen A., Levenberg S, & Spiller K.L. (2020). Macrophages of diverse phenotypes drive vascularization of engineered tissues. Science Advances, 6(18), eaay6391.
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