Each Tet-On 3G inducible gene expression system includes two core elements:
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.
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.
Data: 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.
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).
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.
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.
Click the “Data” tab above to see a comparison of background expression from PTRE3G versus our previous TRE promoter.
Bidirectional vectors contain a specialized TRE3G promoter (PTRE3G-BI) that allows for inducible, simultaneous, and equivalent expression of two transgenes.
Click the “Inducible Coexpression” tab above to learn more about the Bidirectional Tet-On 3G Systems.
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).
Figure 1. 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.
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.
Figure 1. 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.
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.
Figure 1. 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.
Figure 2. 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.
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.
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).
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.
Figure 1. 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.
Figure 1. 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:
