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Products >  Inducible_Systems >  TetSystems_Product_Overview >  Tet-On_3G_Overview

Discover Tet-On 3G Inducible Gene Expression

Choose from the following categories to learn more about Tet-On 3G.

Each Tet-On 3G inducible gene expression system includes two core elements:

  1. The inducible PTRE3G promoter, which provides the lowest basal expression of any tetracycline-inducible promoter in the absence of bound transactivator. PTRE3G retains very strong induced expression when activated by transactivator binding.
  2. The Tet-On 3G transactivator, which binds to and activates expression from TRE promoters in the presence of doxycycline (Dox). Tet-On 3G is optimized for expression in human cells and for sensitivity to Dox.

Why Use Tet-On 3G Systems?

When Tet-On was launched by Clontech in 1996, it was the premier inducible expression system. However, its performance has been surpassed by two subsequent generations of the Tet-On system. Compared to its predecessors, the Tet-On 3G Systems demonstrate lower basal expression and higher sensitivity to the inducer doxycycline (Dox).

  1. Lowest Basal Expression

    The PTRE3G promoter contains mutations that reduce background expression by 5–20 fold compared to our previous tightest promoter, PTight.

    Tetracycline inducible expression, lowest background
  2. Highest Sensitivity

    The Tet-On 3G transactivator contains mutations that significantly increase its sensitivity to Dox. This increased sensitivity is particularly advantageous for in vivo studies in tissues where high Dox concentrations are difficult to attain (e.g., brain).

    Tet-On 3G has the most sensitive transactivator
  3. Tunable Expression

    Adjust expression to the level you need. Simply titrate Dox to the concentration that gives you the level of expression required for your experiment.

    Tet-On 3G dose-dependence to Dox
Download the latest PDF

Want to learn more? Our technical note explains how Tet-On 3G was designed and why it provides the best performance.

The PTRE3G Inducible Promoter: Tetracycline-Inducible Expression, Lowest Basal Activity Level

The tetracycline responsive element (TRE) consists of seven repeats of a 19-bp tet operator sequence located upstream of a minimal CMV promoter. It provides very low basal expression and high maximal expression after induction. Although the tet operator repeats are identical in all Tet-On generations, the junction sequences in the Tet-On 3G version (PTRE3G) have been altered to an even spacing and the central portions have been randomized. These changes minimize background expression, which results in an improved dynamic expression range compared to previous TRE promoters.

PTRE3G provides high maximal expression

Bidirectional PTRE3G Promoter

Bidirectional vectors contain a specialized TRE3G promoter (PTRE3G-BI) that allows for inducible, simultaneous, and equivalent expression of two transgenes.

Download the latest PDF

Want to learn more? Read our sequence comparison of three generations of TRE promoters.

The Tet-On 3G Transcriptional Activator: Highest Sensitivity to Doxycycline

Tet-On 3G is a modified, improved Tet-On Advanced transcriptional activator protein (transactivator) which has been evolved to display far higher sensitivity to doxycycline. Tet-On 3G generates very high maximal expression and responds to lower Dox concentrations than its predecessors. These Dox concentrations are far below cytotoxic levels for either cell culture or transgenic studies. The increased Dox sensitivity is particularly advantageous for in vivo studies in tissues where high Dox concentrations are difficult to attain (e.g., brain).

Doxycycline inducible gene expression

When Dox binds to the Tet-On 3G transactivator, the transactivator undergoes a conformational change that allows it to bind to PTRE3G and initiate transcription.

What is the Tet-On 3G Transactivator?

The transactivator consists of a modified bacterial Tet repressor (TetR) fused to three minimal VP16 activation domains to create a transcriptional activator protein (transactivator). Five amino acid changes convert Clontech’s Tet-On Advanced transactivator to Tet-On 3G.

Download the latest PDF

Want to learn more? Read our sequence comparison of three generations of Tet-On transactivators.

Tet-Inducible Expression in Stem Cells & Hematopoietic Cells: EF-1 Alpha Systems

Some types of cells (particularly hematopoietic cells and stem cells) tend to silence CMV promoters over time. EF-1 alpha promoter versions of the Tet-On 3G systems provide consistent long-term expression of the Tet-On 3G transactivator in these cells, as well as a broad range of other cell types.

Express Tet-On 3G from EF1a in stem cells

The Tet-On 3G Inducible Expression System (EF1alpha Version) provides inducible expression in stem cells. Mouse embryonic stem cells were cotransfected with pTRE3G-ZsGreen1 and pEF1a-Tet3G using Xfect mESC Transfection Reagent. The stem cells show ZsGreen1 expression only in the presence of Dox.

Express Tet-On 3G from EF1a in Jurkat cells

The Tet-On 3G Inducible Expression System (EF1alpha Version) provides inducible expression in hematopoietic cells. Jurkat cells were transfected with Tet3G to pEF1a-Tet3G using Xfect Transfection Reagent, and stable clones were selected by limiting dilution. 83% of the Jurkat Tet-On 3G clones that we tested showed strong inducible expression. Such levels of control were not possible with previous versions of Tet-On for this cell line.

Inducible Coexpression

We offer two types of Tet-inducible gene coexpression. Both types give you a choice of expressing a target gene and a reporter gene, or two target genes.

Bidirectional Tet-On 3G Systems

Bidirectional vectors contain a specialized TRE3G promoter (PTRE3G-BI) that allows for inducible, simultaneous, and equivalent expression of two transgenes. Bidirectional vectors allow you to monitor expression of a target gene indirectly, via co-induced expression of a bright green or red fluorescent protein. Both proteins are expressed equivalently from the bidirectional Tet promoter.

Read more about Bidirectional Tet-On 3G Systems >>

Tetracycline inducible expression using a bidirectional promoter

Doxycycline-regulated expression of two genes. ZsGreen1 and firefly luciferase were cloned into separate multiple cloning sites that flank the inducible bidirectional promoter in the pTRE3G-BI vector. The plasmid was then cotransfected into HEK 293 cells with pCMV-Tet3G (which expresses the Tet-On 3G transactivator). Cells were treated with the indicated doses of doxycycline, and induced gene expression was measured 24 hr later (RLU = Relative Light Units).

Bicistronic Tet-On 3G Systems

Bicistronic (IRES-containing) vectors allow you to express two proteins simultaneously but separately, from the same RNA transcript. Bicistronic vectors are ideal for stable clone selection, because you can monitor expression of your transgene together with a bright fluorescent protein (mCherry or ZsGreen1). Please note that expression of the proteins that are upstream and downstream of the IRES sequence varies.

Read more about Bicistronic Tet-On 3G Systems >>

Monitor co-induced expression with IRES vectors

The Tet System In Vivo

Conditional mouse models are made by crossing two transgenic mouse lines: one expressing the Tet-On transactivator under the control of a tissue-specific promoter, and the other containing the gene of interest cloned behind a TRE promoter. Expression of the gene under study in the double-transgenic progeny is controlled by the presence or absence of Dox in the drinking water.

Tet-inducible mouse models

Hundreds of Tet-transgenic mouse lines are currently available throughout the scientific community or in mouse repositories. Although a Tet-On 3G mouse has not yet been created, we expect that the increased sensitivity of Tet-On 3G will be particularly advantageous for in vivo studies in tissues where high Dox concentrations are difficult to attain (e.g., brain).

Please see the Tet Systems website for a list of Tet-transgenic mouse lines >>

We offer three types of viral delivery for Tet-inducible gene expression:

Lenti-X Tet-On 3G Systems

  • Highest performing inducible lentivirus systems
  • Tightest control of gene expression
  • Each system includes an optimized, 4th generation packaging mix designed to produce high-titer lentivirus.
Lentiviral Tet-On 3G vector map

Read more about Lentiviral Tet-On 3G Systems >>

Retro-X Tet-On 3G System

  • Low background, high induction levels
  • Includes the GP2-293 packaging cell line
  • Choose the best-suited envelope for your target cells (VSV-g, ecotropic, amphotropic or dualtropic)
Retroviral Tet-On 3G vector map

Read more about Retroviral Tet-On 3G Systems >>

Adeno-X Tet-On 3G System

  • All-in-one Tet-inducible vector
  • Tightest control of gene expression
  • Just as easy as plasmid cloning! Clone your transgene directly into the adenoviral genome; no shuttle vector needed
Tetracycline-inducible adenovirus

Read more about Adenoviral Tet-On 3G Systems >>

Tet System Approved Fetal Bovine Serum: Guaranteed Tetracycline-Free

With the greatly increased sensitivity of the Tet-On 3G Systems, it is more important than ever that the serum you use for your studies is tetracycline-free. Only Clontech performs actual inducibility tests on a sensitive Tet-inducible cell line in order to provide an absolute guarantee that your serum is tetracycline-free.

Why is this necessary?

Cattle are routinely fed tetracyclines to guard against infection and build muscle mass. Any residual tetracyclines in serum will increase the basal expression from our highly sensitive Tet-On 3G systems.

Read more about Tet System Approved Serum >>

Tet-free FBS produces consistent results

Only fetal bovine serum from Clontech is functionally tested to guarantee consistent results with tetracycline-inducible expression systems.

Detect Tet-On and Tet-Off Proteins with the TetR Monoclonal Antibody

The TetR Monoclonal Antibody (Clone 9G9) was raised against an essential component of the Tet-On and Tet-Off transactivator proteins. Use the TetR Monoclonal Antibody to simplify the process of developing a Tet-inducible expression system: detect Tet-On 3G, Tet-On Advanced, and Tet-Off Advanced proteins on Western blots.

Read more about TetR Monoclonal Antibody >>

TetR Monoclonal Antibody detects TetR, Tet-On 3G, Tet-On Advanced and Tet-Off Advanced

Western blot detection of the Tet-On Advanced and Tet-Off Advanced transactivators using the TetR Monoclonal Antibody (Clone 9G9).

Note: Antibody-based detection of these tetracycline transactivators in cell lines is not a substitute for a functional test. Some highly inducible clones may express tetracycline transactivators at levels that are undetectable on a Western blot.

Licensing Tet-On 3G Systems

Use of the Tetracycline controllable expression systems (the “Tet Technology”) is covered by a series of patents which are proprietary to TET Systems GmbH & Co. KG. There are specific licensing procedures that apply to for-profit companies and institutions. To determine whether you need a license to place an order, please log in to the Clontech website or contact the Clontech licensing group at 650.919.7320 or licensing@clontech.com.

Tet technology: Precise control of gene expression

Tet-One, Tet-On 3G, and Tet-Express Products

Tet-One Systems (plasmid, retroviral and lentiviral versions available)

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631844 Lenti-X Tet-One Inducible Expression System
631847 Lenti-X Tet-One Inducible Expression System (Puro)
634301 Tet-One Inducible Expression System
634304 Retro-X Tet-One Inducible Expression System
634307 Retro-X Tet-One Inducible Expression System (Puro)

Tet-On 3G Plasmid Systems

Tet-On 3G

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631168 Tet-On 3G Inducible Expression System
631164 Tet-On 3G Inducible Expression System (with ZsGreen1)
631165 Tet-On 3G Inducible Expression System (with mCherry)
631166

Tet-On 3G Inducible Expression System (Bicistronic Version)

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631167

Tet-On 3G Inducible Expression System (EF1alpha Version)

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Tet-On 3G Bidirectional Promoter

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631337 Tet-On 3G Bidirectional Inducible Expression System
631338 Tet-On 3G Bidirectional Inducible Expression System (with mCherry)
631339 Tet-On 3G Bidirectional Inducible Expression System (with ZsGreen1)

Tet-On 3G EF1-Alpha Promoter

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631340 Tet-On 3G Bidirectional Inducible Expression System (EF1alpha Version)
631341 Tet-On 3G Bidirectional Inducible Expression System (EF1alpha, mCherry)
631342 Tet-On 3G Bidirectional Inducible Expression System (EF1alpha, ZsGreen1)
631346 Tet-On 3G Inducible Expression System (EF1alpha, Bicistronic)
631347 Tet-On 3G Inducible Expression System (EF1alpha, mCherry)
631348 Tet-On 3G Inducible Expression System (EF1alpha, ZsGreen1)

Tet-On 3G Lentiviral Systems

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631187 Lenti-X Tet-On 3G Inducible Expression System
631349 Lenti-X Tet-On 3G Inducible Expression System (with mCherry)
631350 Lenti-X Tet-On 3G Inducible Expression System (with ZsGreen1)
631351 Lenti-X Tet-On 3G Inducible Expression System (Bicistronic Version)
631352 Lenti-X Tet-On 3G Inducible Expression System (EF1alpha, mCherry)
631353 Lenti-X Tet-On 3G Inducible Expression System (EF1alpha, ZsGreen1)
631354 Lenti-X Tet-On 3G Inducible Expression System (EF1alpha, Bicistronic)

Tet-On 3G Retroviral System

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631188 Retro-X Tet-On 3G Inducible Expression System

Tet-On 3G Adenoviral System

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631180 Adeno-X Adenoviral System 3 (Tet-On 3G Inducible)

Tet-Express Systems

Tetracycline-Inducible Expression

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631169 Tet-Express Inducible Expression System
631170 Tet-Express Inducible Expression System (Bicistronic Version)
631171 Tet-Express Inducible Expression System (with mCherry)
631172 Tet-Express Inducible Expression System (with ZsGreen1)
631343 Tet-Express Inducible Expression System (Bidirectional Version)
631344 Tet-Express Inducible Expression System (Bidirectional, mCherry)
631345 Tet-Express Inducible Expression System (Bidirectional, ZsGreen1)

Lentiviral Tet-Express

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631189 Lenti-X Tet-Express Inducible Expression System
631355 Lenti-X Tet-Express Inducible Expression System (with mCherry)
631356 Lenti-X Tet-Express Inducible Expression System (with ZsGreen1)
631357 Lenti-X Tet-Express Inducible Expression System (Bicistronic Version)

Retroviral Tet-Express

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631190 Retro-X Tet-Express Inducible Expression System

Tet-On 3G Cell Lines

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Tet System Approved Fetal Bovine Serum

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TetR Monoclonal Antibody

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