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Hela tet off

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The HeLa Tet-Off is a cell line genetically engineered to express a tetracycline-controlled transactivator protein. This system allows for the regulation of gene expression in a tetracycline-dependent manner.

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Lab products found in correlation

Hela tet on, by Takara Bio (2 mentions) Penicillin, by Thermo Fisher Scientific (2 mentions) Streptomycin, by Thermo Fisher Scientific (2 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Porcine gelatin, by Merck Group (1 mentions) Heat inactivated fetal bovine serum, by Thermo Fisher Scientific (1 mentions) Hek293a, by American Type Culture Collection (1 mentions) L glutamine, by Thermo Fisher Scientific (1 mentions) Hek293t, by American Type Culture Collection (1 mentions) Huvecs, by Lonza (1 mentions) Dmem plus glutamax, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions) Hek293t cell, by American Type Culture Collection (1 mentions) Primary human umbilical vein endothelial cells huvecs, by Lonza (1 mentions) Egm 2 medium, by Lonza (1 mentions) Pci neo vector, by Promega (1 mentions) Pcdna5 frt to vector, by Thermo Fisher Scientific (1 mentions) Griptite 293 msr, by Thermo Fisher Scientific (1 mentions) Hela flp in t rex, by Takara Bio (1 mentions)

8 protocols using hela tet off

1

Cell Line Characterization and Treatment

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HeLa Tet-ON (Clontech), HeLa Tet-OFF (Clontech), and A9D GRN patient fibroblasts (ND40082; obtained from NINDS Human Genetics Resource Center DNA and Cell Line Repository; https://www.coriell.org/1/ninds) were used.
For several experiments, HeLa Tet-ON cells were used for continuity, but doxycycline induction was not used. In these cases, we refer to these cells as “HeLa” in the manuscript to avoid confusion. Cell lines have been further specified in Supplemental Experimental Procedures.
Cycloheximide 50 μg/mL was used for 24 hr in HeLa Tet-ON and 10 μg/mL for indicated time in A9D GRN fibroblasts.
Doxycycline 1 μg/mL was used for indicated time in HeLa Tet-OFF.
Pinarbasi E.S., Karamyshev A.L., Tikhonova E.B., Wu I.H., Hudson H, & Thomas P.J. (2018). Pathogenic Signal Sequence Mutations in Progranulin Disrupt SRP Interactions Required for mRNA Stability. Cell reports, 23(10), 2844-2851.
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2

Culturing Human Cell Lines for Research

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Human embryonic kidney 293T and 293A cells (HEK-293T and HEK-293A, respectively; ATCC, Manassas, VA, USA) and human cervical adenocarcinoma cells expressing a tetracycline-inducible repressor (HeLa Tet-Off; Clontech, Mountain View, CA, USA) were cultured in Dulbecco’s modified Eagle medium (DMEM; Gibco, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM l-glutamine (Gibco). Pooled human umbilical vein endothelial cells (HUVECs; Lonza, Basel, Switzerland) were cultured in endothelial cell growth medium 2 (EGM-2; Lonza). For HUVEC passaging, tissue culture plates were precoated for 30 min at 37°C with 0.1% (wt/vol) porcine gelatin (Sigma, St. Louis, Missouri, USA) in 1× phosphate-buffered saline (PBS; Gibco). All cells were routinely tested for mycoplasma by PCR method.
Castle E.L., Robinson C.A., Douglas P., Rinker K.D, & Corcoran J.A. (2021). Viral Manipulation of a Mechanoresponsive Signaling Axis Disassembles Processing Bodies. Molecular and Cellular Biology, 41(11), e00399-21.
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3

HeLa and HEK293 Cell Culture

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HeLa Tet-Off (Clontech) and HEK293 were maintained in DMEM plus Glutamax (Gibco) supplemented with 10% heat-inactivated FBS (Atlanta Biologicals) in a humidified 37°C, 5% CO2 incubator. Cultures were passed every 2–3 days. The HeLa Tet-Off cells were a gift obtained directly from Gerald Wilson (U Maryland, Baltimore). HEK293 cells were obtained from Tazuko Hirai in Bruce Howard's laboratory at the NIH. Our HEK293 and HeLa cell DNAs were both authenticated by the ATCC via STR profiling. HeLa Tet-Off cells are not commonly misidentified cell lines as listed by the International Cell Line Authentication Committee. Standardized mycoplasma testing (ATCC) was performed and tested negative.
Mattijssen S., Arimbasseri A.G., Iben J.R., Gaidamakov S., Lee J., Hafner M, & Maraia R.J. (2017). LARP4 mRNA codon-tRNA match contributes to LARP4 activity for ribosomal protein mRNA poly(A) tail length protection. eLife, 6, e28889.
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4

Cell Culture Protocols for Multiple Cell Lines

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HeLa Tet-Off (Clontech), Phoenix-Amphotropic (a kind gift from G. Nolan, Stanford), and HEK293T cells (ATCC) were maintained at 37°C in a 5% CO2 atmosphere in Dulbecco's modified Eagle's medium containing 100 U of penicillin and streptomycin per ml and 10% heat-inactivated fetal bovine serum. Primary human umbilical vein endothelial cells (HUVECs) were purchased from Lonza. Cultures were expanded in EGM-2 medium (Lonza) on tissue culture plates coated with 0.1% (wt/vol) gelatin (in phosphate-buffered saline [PBS]) and used between passages 5 and 7 for experiments. The BCBL-1 primary effusion lymphoma (PEL) cell line was cultured in RPMI medium containing 10% heat-inactivated fetal bovine serum and 55 µM ß-mercaptoethanol.
Corcoran J.A., Johnston B.P, & McCormick C. (2015). Viral Activation of MK2-hsp27-p115RhoGEF-RhoA Signaling Axis Causes Cytoskeletal Rearrangements, P-body Disruption and ARE-mRNA Stabilization. PLoS Pathogens, 11(1), e1004597.
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5

Plasmid Constructs and Cell Lines

Cited 1 time
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The plasmid constructs β-globin-WT and -PTC39; TPI-WT and -PTC160; TPI-RAB7A, -TNF and -IL6 containing an XRN1-resistant structure from the Murray Valley encephalitis virus were generated by cloning the respective DNA fragments8 (link) into the pcDNA5/FRT/TO vector (Thermo Fisher Scientific). The mVenus and lacZ control plasmid were constructed by inserting the respective DNA fragments into the pCI-neo vector (Promega) as described previously14 (link).
HEK-293 Flp-In T-REx (293 FT; Thermo Fisher Scientific), HeLa Flp-In T-REx (HeLa FT; established by Elena Dobrikova and Matthias Gromeier, Duke University Medical Center), HeLa Tet-Off (Clontech), GripTite 293 MSR (Thermo Fisher Scientific), U2OS and MCF-7 cells were cultured in Dulbecco’s Modified Eagle’s Medium (Gibco, Life Technologies) supplemented with Penicillin-Streptomycin and 9% FBS. The cells were maintained at 37 °C, 5% CO2 and 90% humidity.
Gerbracht J.V., Boehm V, & Gehring N.H. (2017). Plasmid transfection influences the readout of nonsense-mediated mRNA decay reporter assays in human cells. Scientific Reports, 7, 10616.
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6

Culturing Human Cancer Cell Lines

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Human cervical cancer HeLa MDR-Off [23 (link)], HeLa TET-off (Clontech Laboratories, Mountain View, CA) and human embryonic kidney 293T cell lines were cultured in Dulbecco's Modified Eagle's medium (DMEM) in high glucose (4.5 g/l), supplemented with 2 mM L-glutamine, 100 I.U./mL penicillin, 100 μg/mL streptomycin and 10% tetracycline approved FBS (Clontech). The parental KB-3-1 cells (a subclone of HeLa) and their drug-resistant sublines KB-8-5, KB-8-5-11 and KB-C1 were grown with colchicine 10 ng/mL, 100 ng/mL and 1 μg/mL, respectively. All variant sublines were also cultured in DMEM supplemented with 10% FBS, penicillin and streptomycin (Gibco, Grand Island, NY). Cells were cultured at 37 °C with 5% CO2 and relative humidity maintained at 95%. Cell cultures beyond passage 10 were discarded.
Souza P.S., Madigan J.P., Gillet J.P., Kapoor K., Ambudkar S.V., Maia R.C., Gottesman M.M, & Fung K.L. (2015). Expression of the multidrug transporter P-glycoprotein is inversely related to that of apoptosis-associated endogenous TRAIL. Experimental cell research, 336(2), 318-328.
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7

Cell culture conditions and treatments

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In all experiments, HeLa tet-off (Clontech), RAW 264.7 (ATCC) and NIH 3T3 tet-off (Clontech) cells were grown in DMEM (Gibco) with 10% heat-inactivated fetal bovine serum (FBS, Gibco). When indicated, RAW 264.7 cells were treated with 100 ng ml−1 lipopolysaccharide (LPS; Sigma-Aldrich, L6529). NIH 3T3 tet-off cells were synchronized in G0 by growing cells for 48 h in DMEM with 0.2% heat-inactivated FBS, followed by replacement of media with DMEM containing 20% heat-inactivated FBS (Gibco).
Durand S., Franks T.M, & Lykke-Andersen J. (2016). Hyperphosphorylation amplifies UPF1 activity to resolve stalls in nonsense-mediated mRNA decay. Nature Communications, 7, 12434.
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8

Cell Line Characterization and Treatment

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HeLa Tet-ON (Clontech), HeLa Tet-OFF (Clontech), and A9D GRN patient fibroblasts (ND40082; obtained from NINDS Human Genetics Resource Center DNA and Cell Line Repository; https://www.coriell.org/1/ninds) were used.
For several experiments, HeLa Tet-ON cells were used for continuity, but doxycycline induction was not used. In these cases, we refer to these cells as “HeLa” in the manuscript to avoid confusion. Cell lines have been further specified in Supplemental Experimental Procedures.
Cycloheximide 50 μg/mL was used for 24 hr in HeLa Tet-ON and 10 μg/mL for indicated time in A9D GRN fibroblasts.
Doxycycline 1 μg/mL was used for indicated time in HeLa Tet-OFF.
Pinarbasi E.S., Karamyshev A.L., Tikhonova E.B., Wu I.H., Hudson H, & Thomas P.J. (2018). Pathogenic Signal Sequence Mutations in Progranulin Disrupt SRP Interactions Required for mRNA Stability. Cell reports, 23(10), 2844-2851.
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