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Tet on advanced system

Manufactured by Takara Bio
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The Tet-on Advanced System is a tetracycline-inducible gene expression system that allows for tight and reversible control of target gene expression. It consists of a transactivator protein and a response element that work together to induce gene expression upon the addition of tetracycline or its derivatives.

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Lab products found in correlation

Hcc1937, by Takara Bio (3 mentions) Hcc1937, by Nacalai Tesque (3 mentions) Zeocin, by InvivoGen (3 mentions) Rpmi 1640, by Nacalai Tesque (3 mentions) Hcc1937, by American Type Culture Collection (3 mentions) Doxycycline, by Takara Bio (2 mentions) Doxcycline, by Takara Bio (2 mentions) Plvx tight puro, by Takara Bio (1 mentions) Doxycycline, by Merck Group (1 mentions) Rwpe 1, by Thermo Fisher Scientific (1 mentions) Ksf medium, by Thermo Fisher Scientific (1 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions) Rwpe 1, by American Type Culture Collection (1 mentions) Rwpe 2, by American Type Culture Collection (1 mentions) Aps coated slides, by Matsunami (1 mentions) Fetal bovine serum (fbs), by Merck Group (1 mentions)

Close spelling variants of this product

Lenti-TM Tet-On® Advanced Inducible Expression System Lenti-X Tet-On Advanced System Tet-On Advanced Inducible Gene Expression System PLVX Tet-On Advanced plasmid system Lenti-XTM Tet-On® Advanced Inducible Expression System Retro-X™ Tet-On® Advanced Inducible Expression System Lenti-X Tet-On advanced inducible expression system Tet-On Advanced gene expression system Retro-X Tet-On Advanced system Tet-on system

6 protocols using tet on advanced system

1

Generating Inducible CK1 Overexpression Models

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Wild type CK1 isoform cDNA was amplified using the Human Multiple Tissue cDNA (MTC) Panel II (Clontech, Saint-Germain-en-Laye, France) and isoform specific primers. CK1 cDNAs were cloned into the inducible lentiviral vector PLVX-tight-PURO (Clontech) by using In-fusion-HD Liquid Kits (Clontech) according to the manufacturer’s protocol. Sanger-sequencing was performed for verification of the correct cloned cDNA. Lentiviral particles were produced in HEK293T cells using the second-generation packing and envelope plasmids pCMVΔR8.2 and pMD2.G. Cells were transduced with lentiviruses as described previously [16 (link)] and doxycycline inducible melanoma cells were generated according to the manufacturer’s instructions (Tet-on Advanced System, Clontech). For overexpression of CK1α the previously described adenovirus was used [9 (link)].
Sinnberg T., Wang J., Sauer B, & Schittek B. (2016). Casein kinase 1α has a non-redundant and dominant role within the CK1 family in melanoma progression. BMC Cancer, 16, 594.
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2

Inducible Expression of TMEFF2 in Prostate Cell Lines

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The 22Rv1, RWPE1 and RWPE2 cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA). The human prostate epithelial cell line RWPE1 and its Ki-ras transformed tumorigenic derivative, RWPE2, were cultured in KSF medium (Invitrogen, Carlsbad, CA). The human prostate carcinoma cell line 22Rv1 was maintained in RPMI-1640 medium (Invitrogen, Carlsbad, CA). TMEFF2 full length and ΔGA expression constructs were previously described [24 (link), 26 (link)]. Development of a system for inducible expression of FL_TMEFF2 and TMEFF2_ΔGA in RWPE2 cells was achieved using the Clontech’s Tet-On Advanced system (Clontech, Mountain View, CA) essentially as described before for RWPE1 cells [24 (link)]. To inducibly express TMEFF2, cultures were grown in the presence of doxycycline (250 ng/ml; Sigma, St. Louis, MO). 22Rv1 cells transduced with pLKO.1 vectors containing shRNA to TMEFF2 or scramble control were described before [25 (link)].
Chen X., Corbin J.M., Tipton G.J., Yang L.V., Asch A.S, & Ruiz-Echevarría M.J. (2014). THE TMEFF2 TUMOR SUPPRESSOR MODULATES INTEGRIN EXPRESSION, RHOA ACTIVATION AND MIGRATION OF PROSTATE CANCER CELLS. Biochimica et biophysica acta, 1843(6), 1216-1224.
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3

Inducible Wild-type p53 Expression in Triple Negative Breast Cancer Cells

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The human breast cancer cell line HCC1937 was purchased from American Type Culture Collection (ATCC). The HCC1937 cells were negative for expression of ER, PR, and HER2, referred to as a triple negative tumor, and have mutations of TP53 and BRCA1. HCC1937 cells were stably transfected with a wt-p53-inducible plasmid (Tet-on Advanced system, Clontech, USA), and one of the isolated clones was designated as HCC1937/p53 and used for the experiments. The HCC1937/p53 cells were cultured in RPMI1640 (Nacalai tesque, Kyoto, Japan), containing 10% FBS (SIGMA, USA), and ZeocinTM (1 μg/mL, InvivoGen, USA). The HCC1937/p53 cells were cultured on APS-coated slides (MATSUNAMI, Japan) in doxycycline (Takara, 1 ng/mL)—containing media for 1–10 days, and those cells treated with doxcycline for 1 day were designated as dox1d.
Kashii-Magaribuchi K., Takeuchi R., Haisa Y., Sakamoto A., Itoh A., Izawa Y., Isa M., Fukuzawa M., Murakami M, & Takahashi R. (2016). Induced Expression of Cancer Stem Cell Markers ALDH1A3 and Sox-2 in Hierarchical Reconstitution of Apoptosis-resistant Human Breast Cancer Cells. Acta Histochemica et Cytochemica, 49(5), 149-158.
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4

Characterization of Triple-Negative Breast Cancer Cell Line HCC1937

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The human breast cancer cell line HCC1937 was purchased from the American Type Culture Collection (ATCC) [17 (link)]. HCC1937 cells were negative for the expression of ER, PR, and HER2, referred to as a triple-negative tumor, and had nonsense mutations at codon 306 of TP53 and insertion C at nucleotide 5382 (5382C) of the BRCA1 gene. HCC1937 cells had already been stably transfected with a WT-p53-inducible plasmid (Tet-on Advanced System, Clontech, USA), and one of the isolated clones from a single cell was designated as HCC1937/p53 [23 (link), 25 (link)]. HCC1937/p53 cells were cultured in RPMI1640 (Nacalai Tesque, Kyoto, Japan) containing 10% fetal bovine serum (FBS) [22 (link)] (SIGMA, USA) and ZeocinTM (1 μg/mL, InvivoGen, USA).
Inoue S., Imanishi M., Kanzaki A., Fujimoto A., Maeyama M., Okamoto A., Matsuda H., Yoshikawa K, & Takahashi R. (2022). Role of Cancer Stem-like Cells in the Process of Invasion and Mesenchymal Transformation by a Reconstituted Triple-negative Breast Cancer Cell Population Resistant to p53-induced Apoptosis. Acta Histochemica et Cytochemica, 55(5), 169-184.
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5

Establishing Triple-Negative Breast Cancer Model

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The human breast cancer cell line HCC1937 was purchased from American Type Culture Collection (ATCC). The HCC1937 cells were negative for expressions of ER, PR, and HER2, referred to as a triple-negative tumor, and had mutations of TP53 and BRCA1 [24 (link)]. HCC1937 cells were stably transfected with a wt-p53-inducible plasmid (Tet-on Advanced System, Clontech, USA), and one of the isolated clones was designated as HCC1937/p53 and used for the experiments. The HCC1937/p53 cells were cultured in RPMI1640 (Nacalai Tesque, Kyoto, Japan), containing 10% fetal bovine serum (FBS) (SIGMA, USA) [18 (link)], and ZeocinTM (1 μg/mL, InvivoGen, USA). The HCC1937/p53 cells were cultured in doxycycline (Takara, 1 ng/mL)-containing media for 1–7 days, and those cells treated with doxcycline for 2 days were designated as Dox2d [20 (link)].
Izawa Y., Kashii-Magaribuchi K., Yoshida K., Nosaka M., Tsuji N., Yamamoto A., Kuroyanagi K., Tono K., Tanihata M., Imanishi M., Onishi M., Sakiyama M., Inoue S, & Takahashi R. (2018). Stem-like Human Breast Cancer Cells Initiate Vasculogenic Mimicry on Matrigel. Acta Histochemica et Cytochemica, 51(6), 173-183.
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6

Tetracycline-Inducible INCENP Expression

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A system for tetracycline-inducible expression (Tet-On) of INCENP was established using the Tet-On Advanced System (Clontech). Briefly, Tet-On Advanced HeLa-S3 cells were generated by transfection with pTet-On-Advanced vector and selection with 800 μg/ml G418. The transfected Tet-On Advanced HeLa-S3 cells were co-transfected with pTRET6myc-INCENPs (WT, S894A, or S894D) and a linear hygromycin marker, and then screened with 200 μg/ml hygromycin in addition to G418. Each clone was maintained with DMEM containing 5% FBS, 100 μg/ml G418, and 100 μg/ml hygromycin. Exogenous INCENPs were induced by the addition of 2 μg/ml Dox and confirmed by western blotting with anti-Myc antibody (Fig. 9).
Yabuta N., Yoshida K., Mukai S., Kato Y., Torigata K, & Nojima H. (2016). Large tumor suppressors 1 and 2 regulate Aurora-B through phosphorylation of INCENP to ensure completion of cytokinesis. Heliyon, 2(7), e00131.
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