Lenti x tet on 3g inducible expression system

Manufactured by Takara Bio

Sourced in Japan, United States

The Lenti-X Tet-On 3G Inducible Expression System is a laboratory tool designed for the regulated expression of target genes in mammalian cells. It utilizes a third-generation Tet-On system to provide tight control over gene expression, allowing for the induction or repression of gene activity as needed.

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Lab products found in correlation

Tet on 3g inducible expression system, by Takara Bio (2 mentions) Xfect transfection reagent, by Takara Bio (2 mentions) P1400, by Solarbio (1 mentions) Puromycin, by Merck Group (1 mentions) Mocetinostat, by Selleck Chemicals (1 mentions) Plvx tre3g vector, by Takara Bio (1 mentions) 5 azacitidine, by Selleck Chemicals (1 mentions) Gsk126, by Selleck Chemicals (1 mentions) Quickchange 2 xl site directed mutagenesis kit, by Agilent Technologies (1 mentions) Doxycycline, by Merck Group (1 mentions) In fusion cloning kit, by Takara Bio (1 mentions)

Close spelling variants of this product

Tet-On 3G Inducible Expression System Tet-On 3G Expression System Tet-On 3G system Lenti-X expression system Tet-on system Lenti-X

14 protocols using lenti x tet on 3g inducible expression system

Inducible ATRX Expression in U-2 OS and Saos-2 Cells

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U-2 OS and Saos-2 cell lines were obtained from ATCC.
ATRX cDNA was cloned into the Tet-on 3G Inducible Expression System (Clontech) and transfected into using Xfect transfection reagent (Clontech), to generate the U-2 OS^ATRX stable cell line. For Sa-OS2 transient transfections ATRX cDNA was tagged with ZsGreen1 (IRES) using the Lenti-X Tet-on 3G Inducible Expression System (Clontech). Co-transfection of 1 μg pLVX-Tet3G and 4 μg PLVX-TRE3G-ATRX-ZsGreen1 vectors was performed in the presence and absence of 0.4 μg/ml doxycycline for 48 hours using Xfect transfection reagent.

Clynes D., Jelinska C., Xella B., Ayyub H., Scott C., Mitson M., Taylor S., Higgs D.R, & Gibbons R.J. (2015). Suppression of the alternative lengthening of telomere pathway by the chromatin remodelling factor ATRX. Nature Communications, 6, 7538.

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Inducible Expression of HA-Treslin in U2OS Cells

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For TRE3G U2OS HA Treslin cell lines, the Lenti-X Tet-On 3G Inducible Expression System (Clontech Laboratories, Inc, catalog no. 631187) was used according to manufacturer’s protocol (31 (link)). In short, U2OS cells were transduced with Tet-On 3G and TRE3G HA Treslin lentiviruses, and enriched populations were selected with puromycin (2 μg/ml) and G418 (600 μg/ml). Cell lines were cultured in puromycin and G418 throughout the experiments. HA Treslin was induced with 10 ng/ml doxycycline 24 h prior to assay unless otherwise stated. Expression of HA-tagged protein was confirmed by Western blot.

Kelly R.L., Huehls A.M., Venkatachalam A., Huntoon C.J., Machida Y.J, & Karnitz L.M. (2022). Intra-S phase checkpoint kinase Chk1 dissociates replication proteins Treslin and TopBP1 through multiple mechanisms during replication stress. The Journal of Biological Chemistry, 298(4), 101777.

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Inducible KLF17 Overexpression in hESCs

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Dox-inducible overexpression of HA-tagged KLF17 was achieved using the Lenti-X Tet-On 3G Inducible Expression System (Clontech; 631363) following the manufacturer's protocol, and as outlined previously (Wamaitha et al., 2015 (link)). Lentiviral packaging was achieved using 7 μg transgene-containing plasmid and the Lenti-X Packaging Single Shot reagents. Lentiviral supernatant was harvested after 48 h and concentrated by ultracentrifugation. To produce stably transduced cells, hESCs were plated under standard conditions and changed into fresh medium the following morning. Then, 24 h post-plating, 10 μl concentrated virus was added to hESCs for transduction overnight (∼16 h). hESCs were dual selected with 150 μg/ml G418 and 0.5 μg/ml puromycin 48 h post-transduction. For induction of transgene expression, Dox was added to mTeSR1 medium at 1 μg/ml. For the RNA-seq experiments, KLF17-inducible hESCs were plated as normal and induction initiated after 24 h by addition of 1 μg/ml Dox to the culture medium (mTeSR1). At ∼30 h, a day 0 (pre-induction) control sample was collected, then both induced (+Dox) and UI samples were collected at 24 h intervals from 48 h (day 1 post-induction) until 144 h (day 5 post-induction). RNA was extracted from the samples and subjected to bulk RNA-seq.

Lea R.A., McCarthy A., Boeing S., Fallesen T., Elder K., Snell P., Christie L., Adkins S., Shaikly V., Taranissi M, & Niakan K.K. (2021). KLF17 promotes human naïve pluripotency but is not required for its establishment. Development (Cambridge, England), 148(22), dev199378.

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Establishing 3×Flag-Tas-Overexpressing Cell Lines

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HeLa and 293TN cells were maintained in DMEM (12,100, Solarbio, China) supplemented with 10% FBS (CP17-1616, Capricorn, Germany) and 1% penicillin/streptomycin (P1400, Solarbio, China) in 5% CO₂ at 37°C. For stable 3˟Flag-Tas-overexpressing cell-line establishment, a Lenti-X™ Tet-On® 3 G Inducible Expression System (Clontech® Laboratories) was used following the manufacturer’s instructions. For knocking out PPM1E, sgRNA (GGGCCAAGCTGTTGAACTA) using the Lentiviral Crispr Toolbox released by ZhangLab was subcloned into LentiCRISPRv2 and then cotransfected with pMD2.0 and psPAX2. Seventy-two hours after transfection, the supernatant of lentiviruses was collected and used to infect HeLa cells for 48 h. Finally, the obtained stable cell lines in which PPM1E was knocked out were selected by puromycin resistance screening in 1 µg/ml for 7 days.

Jie W., Rui-Fen Z., Zhong-Xiang H., Yan W., Wei-Na L., Yong-Ping M., Jing S., Jing-Yi C., Wan-Hong L., Xiao-Hua H., Zhi L, & Yan S. (2022). Inhibition of cell proliferation by Tas of foamy viruses through cell cycle arrest or apoptosis underlines the different mechanisms of virus–host interactions. Virulence, 13(1), 342-354.

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Generating miRNA Screening Vectors

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MicroRNA screen vectors were generated by PCR amplification and subcloned into pLenti‐m3‐blasticidin vector. TP63‐3′ UTR sequence was amplified by genomic PCR using primer pair TP63‐3′ UTR‐forward (F) and TP63‐3′ UTR‐reverse (R), and then cloned into pmirGLO Dual‐Luciferase reporter to construct TP63‐3′ UTR‐wild type (WT) vector. Vectors with deletion of the predicted binding site of miR‐522‐3p in TP63‐3′ UTR, namely, TP63‐3′ UTR‐∆1‐4, were constructed using primer pairs: 522DL1‐F and 522DL1‐R, 522DL2‐F and 522DL2‐R, 522DL3‐F and 522DL3‐R, and 522DL4‐F and 522DL4‐R, respectively. To construct the vector with inducible expression of miR‐522, we amplified genomic DNA using primer 522TRE‐F and 522TRE‐R and cloned into pLVX‐TRE3G vector of Lenti‐X™ Tet‐On 3G Inducible Expression System (631187; Clontech, Beijing, China). All primers are listed in Table 1. ΔNp63α expression vector was kept in our laboratory [6].

Dong Y., Long J., Luo X., Xie G., Xiao Z.J, & Tong Y. (2021). Targeting of ΔNp63α by miR‐522 promotes the migration of breast epithelial cells. FEBS Open Bio, 11(2), 468-481.

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Inducible PRDM10 Overexpression Cell Lines

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To create inducible cell lines that overexpress PRDM10^WT (293 T) and PRDM10^Cys677Tyr (293 T and RPTEC/TERT1), lentiviral production and transduction took place according to the Lenti-X Tet-On 3G Inducible Expression System (Clontech, Takara Bio, Japan) technical manual. In short, PRDM10^WT and PRDM10^Cys677Tyr cDNA were cloned into the pLVX-Tre3G plasmid where after Tre3G-Cas9 and Tet3G lentiviral particles were produced in 293 T cells. For transduction, cells were seeded in a 6-wells plate. The next day growth media was changed for 1 ml media containing viruses. Cells were incubated overnight and after 24 hours media was replaced with 2 ml fresh media. The next day cells were transferred to 10 cm plates and Puromycin (3 μg/ml, Sigma-Aldrich, St. Louis, MO, USA) was added to select for successfully transduced cells.

van de Beek I., Glykofridis I.E., Oosterwijk J.C., van den Akker P.C., Diercks G.F., Bolling M.C., Waisfisz Q., Mensenkamp A.R., Balk J.A., Zwart R., Postma A.V., Meijers-Heijboer H.E., van Moorselaar R.J., Wolthuis R.M, & Houweling A.C. (2022). PRDM10 directs FLCN expression in a novel disorder overlapping with Birt–Hogg–Dubé syndrome and familial lipomatosis. Human Molecular Genetics, 32(7), 1223-1235.

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Overexpression of GRHL2 and Epigenetic Modulations

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Lenti-X Tet-On 3G-Inducible Expression System (Clontech) was used for GRHL2 overexpression. The complementary DNA (cDNA) of GRHL2 was cloned into pLVX-TRE3G vector (631191, Clontech) using standard molecular cloning techniques. The mutated GRHL2* (resistant to shGRHL2 #12) was generated by introducing four silent point mutations to the wild-type cDNA using QuickChangeII XL Site-Directed Mutagenesis Kit (Agilent). The 293T cells were transfected with pLVX-Tet3G, viral packaging mix, and pLVX-TRE3G-GRHL2 or pLVX-TRE3G-GRHL2* using Lenti-X HTX Packaging Mix 2 System (631260, Clontech). Viruses were harvested to infect IOSE523, HeyA8, and OVCA429 shGRHL2 #12 cells. GRHL2 expression was induced by doxycycline (1 μg/ml for 48 or 96 h). Cells were treated with GSK126 (S7061, Selleck Chemicals) at a final concentration of 5 μM for 72 h; mocetinostat (S1122, Selleck Chemicals) at 1 μM (OVCA429 shGRHL2 Tet-GRHL2*) or 0.5 μM (IOSE523 and HeyA8) for 48 h; 5-azacitidine (S1782, Selleck Chemicals) at 1 μM for 144 h.

Chung V.Y., Tan T.Z., Ye J., Huang R.L., Lai H.C., Kappei D., Wollmann H., Guccione E, & Huang R.Y. (2019). The role of GRHL2 and epigenetic remodeling in epithelial–mesenchymal plasticity in ovarian cancer cells. Communications Biology, 2, 272.

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Inducible EGFR-GFP Expression in MDCK and MCF12A Cells

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MDCK II cells were double transfected using Clontech Lenti-X Tet-On 3G Inducible Expression System. A pLVX-Tet3G (Cat# 631187) plasmid was transfected into parental MDCK II cells by combining pLVX-Tet3G plasmid with Lipofectamine 2000 in transfection media (MEM +10% FBS +1% P/S). Selection was established and maintained with 0.5mg/mL G418. Once a stable stock was generated the second plasmid pLVX-Tre3G (either an empty vector (EV), EGFR-GFP^WT, or EGFR-GFP^P667A) was introduced using the same method. Selection of double transfected cells was established and maintained with 3μg/mL puro in addition to 0.5mg/mL G418. MCF12A cells were transiently transfected using a CMV-EGFR-GFP^WT or CMV-EGFR-GFP^P667A plasmid with Lipofectamine 2000 and fixed within 48 hours for imaging.

Greenwood E., Maisel S., Ebertz D., Russ A., Pandey R, & Schroeder J. (2016). Llgl1 prevents metaplastic survival driven by epidermal growth factor dependent migration. Oncotarget, 7(38), 60776-60792.

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Conditional Overexpression of Tlk1 using Tet-On System

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To conditionally overexpress Tlk1, Lenti-X Tet-On 3 G inducible expression system was used following manufacturer’s protocol (Cat#631353; Clontech). Briefly, wild type Tlk1 and D607A mutant were cloned into pLVX-TRE3G-ZsGreen1 vector. To produce virus particle, 293FT cells were transfected with 7 µg of pLVX-TRE3G vector containing Tlk1 or pLVX-Tet3G vector (regulator), 2.25 µg of pMD2.G and 6.75 µg of psPAX2 using Lipofectamine 2000 transfection reagent. Cells were first infected with regulator virus and selected with G-418. And then pLVX-TRE3G-Tlk1 (WT or D607A) virus infected the cells harboring regulator and the cells was selected by G-418 and puromycin. To induce expression of genes, doxycycline (final concentration 100 ng/ml; Cat. #D9891; Sigma-Aldrich) was treated.

Lee J., Kim M.S., Park S.H, & Jang Y.K. (2018). Tousled-like kinase 1 is a negative regulator of core transcription factors in murine embryonic stem cells. Scientific Reports, 8, 334.

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Inducible ATRX Expression in U-2 OS and Sa-OS2 Cells

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U-2 OS and Saos-2 cell lines were obtained from ATCC.
ATRX cDNA was cloned into the Tet-on 3G Inducible Expression System (Clontech) and transfected into using Xfect transfection reagent (Clontech), to generate the U-2 OS^ATRX stable cell line. For Sa-OS2 transient transfections ATRX cDNA was tagged with ZsGreen1 (IRES) using the Lenti-X Tet-on 3G Inducible Expression System (Clontech). Co-transfection of 1 μg·pLVX-Tet3G and 4 μg PLVX-TRE3G-ATRX-ZsGreen1 vectors was performed in the presence and absence of 0.4 μg ml⁻¹ doxycycline for 48 h using Xfect transfection reagent.

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