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Hek293t lentix cells

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HEK293T LentiX cells are a well-characterized human embryonic kidney cell line that is commonly used for the production of lentiviral vectors. These cells are optimized for high-titer lentivirus production and are suitable for a variety of applications, including gene delivery, gene expression, and cell line development.

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Lab products found in correlation

Polyethylenimine (pei), by Polysciences (7 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions) Pen strep, by Thermo Fisher Scientific (2 mentions) Recombinant human hepatocyte growth factor, by Thermo Fisher Scientific (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Glutamax, by Thermo Fisher Scientific (2 mentions) Medium 199, by Thermo Fisher Scientific (2 mentions) Hiv p24 antigen test, by Roche (2 mentions) Plenti 3 ubc luc2, by Applied Biological Materials (2 mentions) Puromycin, by InvivoGen (2 mentions) Penicillin, by Thermo Fisher Scientific (2 mentions) Streptomycin, by Thermo Fisher Scientific (2 mentions) Dulbecco s modified, by Thermo Fisher Scientific (1 mentions) Plenti cmv gfp puro, by Addgene (1 mentions) Puromycin, by Thermo Fisher Scientific (1 mentions) Prsv rev, by Addgene (1 mentions) Lenti x concentrator, by Takara Bio (1 mentions) Gentamycin, by Merck Group (1 mentions) Calphos mammalian transfection kit, by Takara Bio (1 mentions) Sw28 swinging bucket rotor, by Beckman Coulter (1 mentions)

20 protocols using hek293t lentix cells

1

Myoblast Culture from HML Patients

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Myoblasts from the tibialis anterior of two HML patients (P1, P2) and a healthy control (C) were cultured in 4 volumes of Dulbecco’s modified essential medium (DMEM)(Gibco, Waltham, MA, USA) to 1 volume Medium 199 (Gibco) supplemented with 20% FBS (Gibco), 5 ng/ml recombinant human hepatocyte growth factor (Invitrogen, Waltham, MA, USA) and 50 μg/ml gentamycin. HEK 293T Lenti-X cells (Clontech, Mountain View, CA, USA) were cultured in DMEM (Gibco) supplemented with 1% Glutamax (Gibco), 10% FBS and 1% Pen Strep (Gibco). All cells were grown at 37°C with 5% CO2. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated. Studies including humans, where written informed consent was obtained from all participants, were approved by the Regional Ethics Committee for Medical Research at Umeå University (09–105M).
Rawcliffe D.F., Österman L., Lindsten H, & Holmberg M. (2016). The High Level of Aberrant Splicing of ISCU in Slow-Twitch Muscle May Involve the Splicing Factor SRSF3. PLoS ONE, 11(10), e0165453.
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2

Lentiviral Production and Selection

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Lentivirus was produced by PEI (Polysciences Inc.) transfection of HEK293T LentiX cells (Clontech) with gene delivery vector co-transfected with packaging vectors pspax2 and pMD2.G as previously described23 . Supernatants were harvested 72h post-transfection and centrifuged at 20,000 rpm for 2h at 4°C. Virus containing pellets were resuspended in PBS and placed on cells dropwise. Selection of lentivirally-infected cells was achieved with either blasticydin or puromycin, both used at 2μg/ml.
Nakayama R.T., Pulice J.L., Valencia A.M., McBride M.J., McKenzie Z.M., Gillespie M.A., Ku W.L., Teng M., Cui K., Williams R.T., Cassel S.H., Qing H., Widmer C.J., Demetri G.D., Irizarry R.A., Zhao K., J.e.f.f.R.a.n.i.s.h, & Kadoch C. (2017). SMARCB1 is required for widespread BAF complex-mediated activation of enhancers and bivalent promoters. Nature genetics, 49(11), 1613-1623.
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3

Generating Luciferase-Expressing Cell Lines

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Lentivirale particles were generated by co-transfection of HEK293T Lenti-X cells (Clontech) with packaging plasmids (pMD2.G, pMDLg/pRRE, pRSV-REV, Addgen) and the lentiviral transfervector (pLenti-III-UbC-Luc2, Applied Biological Materials Inc.) expressing Luciferase 2. After 48 and 72 hours the supernatants were pooled, filtered through a 45 um filter and ultracentrifuged at 87076 g 4°C for 2 h. The virus titers were determined by HIV p24 antigen test (Elecsys, Roche Diagnostics). MEB-Med-8A cells were transduced with virus particles (MOI 5) and thereafter selected with puromycin and validated for homogenous luciferase activity (Invitrogen).
Ehrhardt M., Craveiro R.B., Holst M.I., Pietsch T, & Dilloo D. (2014). The PI3K inhibitor GDC-0941 displays promising in vitro and in vivo efficacy for targeted medulloblastoma therapy. Oncotarget, 6(2), 802-813.
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4

Lentiviral Transduction and Selection

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Lentivirus was produced by PEI (Polysciences Inc.) transfection of HEK-293-T LentiX cells (Clontech) with gene delivery vector co-transfected with packaging vectors pspax2 and pMD2.G as previously described(Kadoch and Crabtree, 2013 (link)). Supernatants were harvested 72h post-transfection and centrifuged at 20,000 rpm for 2h at 4°C. Virus containing pellets were resuspended in PBS and placed on cells dropwise. Selection of lentivirally-infected cells was achieved with either blasticydin or puromycin, both used at 2μg/ml. Overexpression or knock-down (KD) efficiency was determined by Western blot analyses or RT-qPCR. shRNA constructs used are found in Table S3.
Sandoval G.J., Pulice J.L., Pakula H., Schenone M.A., Takeda D.Y., Pop M., Boulay G., Williamson K.E., McBride M.J., Pan J., Pierre R.S., Hartman E., Garraway L.A., Carr S.A., Rivera M.N., Li Z., Ronco L., Hahn W.C, & Kadoch C. (2018). Binding of TMPRSS2-ERG to BAF chromatin remodeling complexes mediates prostate oncogenesis. Molecular cell, 71(4), 554-566.e7.
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5

Lentiviral Transduction and Selection

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Lentivirus was produced by PEI (Polysciences) transfection of HEK293T LentiX cells (Clontech) with gene delivery vector co-transfected with packaging vectors pspax2 and pMD2.G as previously described (Kadoch and Crabtree, 2015 (link)). Viral supernatants were harvested 72 hr post-transfection and concentrated by ultracentrifugation at 20,000 rpm for 2.5 hr at 4°C. Virus containing pellets were resuspended in PBS and added dropwise to cells in the presence of 10 μg/mL polybrene. Selection of lentivirally-infected cells was achieved with puromycin used at 2 μg/ml. Knockdown efficiency and overexpression was measured by western blot analysis.
Zullow H.J., Sankar A., Ingram D.R., Guerra D.D., D’Avino A.R., Collings C.K., Segura R.N., Yang W.L., Liang Y., Qi J., Lazar A, & Kadoch C. (2022). The FUS::DDIT3 fusion oncoprotein inhibits BAF complex targeting and activity in myxoid liposarcoma. Molecular cell, 82(9), 1737-1750.e8.
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6

Overexpression of Braf Oncogene Variants

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Human BRAFP1 was cloned into pLenti-CMV-GFP-Puro (Addgene 25873) and pCDNA3, and mouse Braf-rs1 was cloned into pCCL.sin.PPT.hPGK.GFP.Wpre (L277, L. Naldini) or pCDNA3-neo. pMSCV-tTA (Addgene #18783) was used to induce Braf-rs1 expression in TRE-BPS MEFs. Lipofectamine 2000 was used for plasmid transfection. Lentivirus or retrovirus was produced in HEK293T LentiX cells (Clontech) co-transfected with VSVG, pMDL, and Rev or Eco helper plasmids, respectively. Viral supernatants were filtered and cells infected in the presence of 5μg/mL polybrene.
Karreth F.A., Reschke M., Ruocco A., Ng C., Chapuy B., Léopold V., Sjoberg M., Keane T.M., Verma A., Ala U., Tay Y., Wu D., Seitzer N., Del Castillo Velasco-Herrera M., Bothmer A., Fung J., Langellotto F., Rodig S.J., Elemento O., Shipp M.A., Adams D.J., Chiarle R, & Pandolfi P.P. (2015). The BRAF pseudogene functions as a competitive endogenous RNA and induces lymphoma in vivo. Cell, 161(2), 319-332.
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7

Lentiviral Luciferase Reporter Generation

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Lentivirale particle were generated by co-transfection of HEK293T Lenti-X cells (Clontech) with packaging plasmids (pMD2.G, pMDLg/pRRE, pRSV-REV, Addgene) and the lentiviral transfervector (pLenti-III-UbC-Luc2, Applied Biological Materials Inc.) expressing Luciferase 2. After 48 and 72 hours the supernatants were pooled, filtered through a 45 um filter and ultra centrifuged at 32000 rpm 4°C for 1h. The virus titers were determined by HIV p24 antigen test (Elecsys, Roche Diagnostics GmbH). MEB-Med-8A cells were transduced with virus particles (MOI 5) and thereafter selected with puromycin for cell clones that show stable luciferase expression (Invitrogen).
Craveiro R.B., Ehrhardt M., Holst M.I., Pietsch T, & Dilloo D. (2014). In comparative analysis of multi-kinase inhibitors for targeted medulloblastoma therapy pazopanib exhibits promising in vitro and in vivo efficacy. Oncotarget, 5(16), 7149-7161.
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8

Lentiviral Production and Selection

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Lentivirus was produced by PEI (Polysciences Inc.) transfection of HEK293T LentiX cells (Clontech) with gene delivery vector co-transfected with packaging vectors pspax2 and pMD2.G as previously described23 . Supernatants were harvested 72h post-transfection and centrifuged at 20,000 rpm for 2h at 4°C. Virus containing pellets were resuspended in PBS and placed on cells dropwise. Selection of lentivirally-infected cells was achieved with either blasticydin or puromycin, both used at 2μg/ml.
Nakayama R.T., Pulice J.L., Valencia A.M., McBride M.J., McKenzie Z.M., Gillespie M.A., Ku W.L., Teng M., Cui K., Williams R.T., Cassel S.H., Qing H., Widmer C.J., Demetri G.D., Irizarry R.A., Zhao K., J.e.f.f.R.a.n.i.s.h, & Kadoch C. (2017). SMARCB1 is required for widespread BAF complex-mediated activation of enhancers and bivalent promoters. Nature genetics, 49(11), 1613-1623.
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9

Efficient Lentiviral Transduction for Stable Gene Delivery

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Lentivirus production was obtained from PEI (Polysciences) transfection of HEK293T LentiX cells (Clontech) with co-transfection of the packaging vectors pspax2 and pMD2.G along with the gene delivery vector. Viral supernatants were collected 72 hours after transfection, underwent ultracentrifugation at 20,000 rpm for 2.5 hr at 4°C to concentrate, and then virus pellets were resuspended in PBS. For infection, the viral pellets were added to cells in a drop wise manner in the presence of polybrene (10 μg/mL). After 48 hours, the media containing the lentivirus was replaced and infected cells were selected by addition of puromycin (2 μg/mL) or blasticidin (10 µg/mL).
McBride M.J., Mashtalir N., Winter E.B., Dao H.T., Filipovski M., D’Avino A.R., Seo H.S., Umbreit N.T., St. Pierre R., Valencia A.M., Qian K., Zullow H.J., Jaffe J.D., Dhe-Paganon S., Muir T.W, & Kadoch C. (2020). The nucleosome acidic patch and H2A ubiquitination underlie mSWI/SNF recruitment in synovial sarcoma. Nature Structural & Molecular Biology, 27(9), 836-845.
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10

Lentiviral Transduction of Cell Lines

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Lentivirus was produced in HEK293T LentiX cells (Clontech) by LT1 (Mirus Bio) transfection with gene delivery vector and packaging vectors GAG/POL and VSV plasmids.17 (link) Viral supernatants were collected 72 h after transfection and concentrated using the LentiX concentrator (Clontech). Virus containing pellets were resuspended in PBS and added dropwise on cells in presence of growth media supplemented with six ug/ml polybrene. Cells infected with lentivirus were selected using puromycin (Invivogen) at a concentration of one ug/ml for SKNMC, EW7, CHP100, HEK293T, HeLa and U2OS or two ug/ml for A673 and MRC5 in the growth medium. MSCs were selected with 0.75 ug/ml puromycin. Overexpression efficiency was determined by immunoblot analysis.
Tak Y.E., Boulay G., Lee L., Iyer S., Perry N.T., Schultz H.T., Garcia S.P., Broye L., Horng J.E., Rengarajan S., Naigles B., Volorio A., Sander J.D., Gong J., Riggi N., Joung J.K, & Rivera M.N. (2022). Genome-wide functional perturbation of human microsatellite repeats using engineered zinc finger transcription factors. Cell Genomics, 2(4), 100119.
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