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Gain Control: The Tet-On/Tet-Off Inducible Expression System

Posted in: Protein Expression and Analysis
Gain Control: The Tet-On/Tet-Off Inducible Expression System

While overexpressing a gene of interest can provide a look into its role in a cell, sometimes it is necessary to control the expression of a gene. You may want to dictate the timing of the protein’s expression or lower its expression level to adequately understand its function. This is particularly relevant when studying genes that may be toxic to your model organism or cell line.

The answer: tetracycline inducible systems

A commonly used inducible mammalian expression system is the Tetracycline-controlled Transcriptional Activation system developed in 1992 by Professors Hermann Bujard and Manfred Gossen at the University of Heidelberg1.

With the “Tet-Off” and “Tet-On” systems, the expression of a gene of interest, or even a short hairpin RNA, can be controlled by the presence (or absence) of tetracycline or one of its derivatives (such as doxycycline).

How it works

Tetracycline systems rely on two elements:

  • A transactivator protein and
  • The activator protein’s consensus binding sites

The activator is the tetracycline transactivator fusion protein (tTA): a “frankenprotein” composed of tetracycline repressor (TetR) and the activation domain of VP16, a transcriptional activator produced by herpes simplex viruses. This fusion protein specifically binds to a tetracyline response element (TRE), a series of repeated tetracycline operator (TetO) sequences located upstream of the promoter of a gene of interest. Binding of tTA to the sequences upstream of the gene of interest increases the transcription of the gene.


In the presence of tetracycline, tTA is unable to bind to the TetO and expression of the gene is turned off (hence the name “Tet-Off”).


In an opposite manner, the “Tet-On” system requires tetracycline in order for tTA to bind to the TRE and induce gene expression. Addition of tetracycline to a Tet-On system causes a conformational change in tTA allowing it to bind to the TRE and turn on the expression of the gene of interest.

Benefits of tet inducible systems

The tetracycline inducible system has many benefits that allow the study of proteins in eukaryotic cells in a controlled manner. It is often the preferred inducible system because it allows for rapid and reversible gene expression. This technology has also been adapted for use in animal model systems where tTA is expressed in specific tissues allowing gene function to be studied in desired cell types.

Drawbacks to tet inducible systems

As with any biotechnology, the tetracycline system is not without its downsides. Often times “leakiness” can be observed in which the gene of interest is expressed even without the addition of tetracycline. Means of reducing this leakiness include recruiting stronger repressors (in the case of Tet-Off systems), or decreasing the stability of the mRNA of your gene of interest through the introduction of AU-rich mRNA destabilizing elements (AREs) in the 3’ UTR region of the gene construct.

Also, tetracycline is an antibiotic that binds to the 30S subunit of bacterial ribosomes. Unfortunately, it can also bind to the 30S subunit of mammalian ribosomes and cause toxicity to mammalian cells when used at high concentrations. You will need to titrate the amount of tetracycline you use to find a balance between cell toxicity and the efficiency of gene expression.

It’s not always about tet

If tet inducible systems don’t work for you, all is not lost. There are other systems out there.

Analogous to the tetracycline system is the LacSwitch system which uses the Lac repressor in place of tTA and isopropyl ß-D-thiogalactopyranoside (IPTG) as an inducible means of inhibiting the Lac repressor.

Other inducible systems that are widely used rely are Cre recombinase, an enzyme capable of removing DNA that lies between DNA recognition sequences called loxP sites, or FLP-FRT recombination, a system similar to Cre recombinase that also uses site-specific recombination of DNA. Unfortunately, these recombination-based systems permanently remove DNA sequences causing irreversible changes to the expression of a gene of interest.

The most appropriate method of gene expression depends on your specific experimental goals. Regardless of which technique you find best suited to your needs, rapid and controllable gene expression can provide a unique glimpse into the function and dynamics of your gene of interest.


1.  Gossen M. and Bujard H. (1992) Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci 15;89(12):5547-5551.



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