Jump to content
Powered by

TET Systems: Controlled gene expression in eukaryotes

The most common and most successful system used for the experimental regulation of gene expression in eukaryotes is based on a gene switch that regulates the resistance of bacteria to tetracycline. The Tet technology also provides researchers with a tool that enables them to specifically, quantitatively and reversibly control the activity of individual genes in vivo and in vitro. The Heidelberg-based company TET Systems sells licences for the Tet technology that, since its inception, has been modified and optimised for a broad range of different functions.

In order to understand the function of a gene in a cell or organism, it has to be possible to experimentally control its expression. The Tet technology, which is based on a genetic circuit that regulates bacterial resistance to the antibiotic tetracycline, is the most frequently used experimental system for the regulation of gene expression in eukaryotes.

TET Systems

The Tet technology, developed at the beginning of the 1990s by Hermann Bujard and his colleagues at the Centre of Molecular Biology in Heidelberg (ZMBH), has been adapted and optimised to a degree that the bacterial control system is also able to switch on and off genes in eukaryotes, including cell culture systems (including human tumour cell lines and pluripotent stem cells) and organisms (from unicellular fungi and parasites such as plasmodia and toxoplasmas to nematodes, insects, plants, fish, amphibians and mammals, in particular mice). The Tet technology enables a highly effective, precise and reversible control of the point in time as well as the degree of gene expression in eukaryotic systems. Its huge potential for application is illustrated by the more than 7,000 publications in leading journals worldwide.

The company TET Systems GmbH & Co. KG, which was founded by Bujard and his colleagues in Heidelberg, is the sole owner of the Tet technology that is protected by a broad range of patents. TET Systems issues licences for the Tet technology and its components, including licences that permit modification and improvement of the system. Academic institutions receive the licence free of charge when they purchase the Tet technology components, on the condition that it is only used for academic research.

Dr. Ernst Boehnlein © TET Systems

Projects paid for by profit-oriented institutions are not eligible for a free-of-charge licence, explains Dr. Ernst Boehnlein, CEO of TET Systems. In addition, the sale or other dissemination of Tet components to third parties is prohibited.

TET Systems offers commercial licences or non-exclusive research licences to companies and other for-profit organisations that want to make use of the technology. The licensing agreements are adapted to the individual requirements of the licensees. TET Systems has a broad network of licensing partners from whom components of the Tet technology and related products and services can be purchased. Clontech (now part of Takara Bio), the preferred partner of TET Systems, has been marketing and selling the Tet technology for more than 15 years.

From a bacterial to a eukaryotic control system

Doxycyline formula

The transcription of a gene that is cloned into a eukaryotic system can only be initiated when placed under the control of a promoter that contains a polylinker for the gene whose activity is to be specifically regulated. Nowadays, the most commonly used effector substance is the tetracycline derivative doxycycline, whose chemical and physiological properties are well understood on the molecular level. Doxycycline has the advantage that it can easily diffuse into almost all cells and is even able to pass through the blood-brain barrier and the placenta. It is highly effective against intracellular pathogens and is not toxic for eukaryotic cells in the concentrations used. Three nanogrammes of doxycycline per cubic centimetre are sufficient to switch a particular gene on or off. The modification of the doxycycline concentration enables the gradual regulation of gene activity over five orders of magnitude without interference with the host cell physiology.

 

Since its first presentation, the Tet System® has engendered numerous modifications for a broad range of applications.  It is based on the tetracycline resistance operon of E. coli whose transcription is inhibited by the Tet repressor in the absence of tetracycline and is induced upon the binding of the antibiotic to the repressor. Bujard and his colleagues fused the Tet repressor with the VP16 activation domain of Herpes simplex, which transformed the repressor into a transcription activator. The hybrid protein is referred to as tetracycline-dependent transactivator (tTA) and is encoded by one of the two vectors of the Tet expression system. The second vector has a multiple cloning site for the target gene to be cloned, whose activity is controlled by tetracycline. The absence of tetracycline or doxycycline leads to the transcription of the gene, whereas the addition of tetracycline or doxycycline prevents the transcription of the gene (Tet-Off variant). Generally, a Tet-On variant is used for eukaryotic applications, where the gene is only transcribed following the addition of doxycycline. In this case, a reverse tetracycline-controlled transactivator (rtTA) was created by the introduction of mutations into the hybrid protein consisting of Tet repressor and viral activation domain. The rtTA only induces the transcription of the gene in the presence of doxycycline.

The ability to use the Tet technology to truly create "conditional mutants", i.e. cells or whole organisms in which the mutation only exerts its effect upon the addition of tetracycline or doxycycline (Tet-On switch) or in its absence (Tet-Off switch), opens up a broad range of possibilities for the analysis of gene functions and for the development of new animal models for human diseases. "Genes can now not only be reversibly switched on and off in a cell type-specific and temporally defined way; an intermediary gene activity that corresponds more to the pathological situation in humans than the "all or nothing" switch can also be selected", said Dr. Sabine Freundlieb, Manager of Scientific Affairs of TET Systems and a former colleague of Bujard at the ZMBH.

Tet technology to combat malaria

Aedes aegypti larvae, modified with the RIDL system and a fluorescence marker. © Oxitec UK

The British biotech company Oxitec (Oxford Insect Technologies) used the Tet switch to develop a smart method to fight off disease-transmitting mosquitoes. This method, known as RIDL® technology, was first used against Aedes mosquitoes, which transmit dengue fever and yellow fever, two diseases caused by arboviruses in tropical countries, and that affect more than 100 million people every year. Since then, the RIDL® technology has also been used to control Anopheles mosquitoes, which transmit malaria. RIDL insects carry a transgene with a Tet switch that is passed on to their offspring and leads to the death of the larvae. When RIDL males are released to mate with female insects in the wild, their progeny inherit the RIDL gene and do not survive to adulthood. The Tet System causes the lethality gene to become conditional; it can be switched off to breed large numbers of insects.

Tet mice as models

In the meantime, around 300 different transgenic Tet mouse lines have been described and many are available for academic research from institutions such as The Jackson Laboratory in the USA, RIKEN in Japan and EMMA (the European Mouse Mutant Archive) in Europe. Mouse models like these can be used to investigate molecular events in the development and progression of a given disease as well as its reversibility and regression; genes and gene products can be identified as pharmacological targets. Moreover, the mice are also excellently suited for preclinical ADME/Tox studies. Conditional mouse models, in which the expression of oncogenes such as c-myc, ras 12 or ErbB2 were strictly controlled by Tet, have led to unexpected findings relating to the mechanisms that control the initiation, progression and regression of tumours. These findings suggest that many tumours depend on the expression pattern triggered by the tumour-inducing oncogene both with regard to their development as well as their further growth. This phenomenon is referred to as constitutive oncogene addiction. In a mouse line that serves as model of Huntington’s disease in humans, a neurodegenerative genetic disease for which no cure exists, the disease-causing mutant of a chimerical mouse/human Huntingtin gene was placed under the control of Tet. The typical neuropathological changes that occurred upon the expression of the Huntingtin gene and that led to severe progressive muscular dystrophy could be reversed by switching off the gene. This spectacular finding gives rise to hopes that it will be possible in future to develop causal therapies even for severe, supposedly progressive diseases. Of course, the somatic gene therapy of diseases requires reliable gene switches such as those provided by the Tet technology.

A new approach for the treatment of diabetes

A recently published study reported that in transgenic mice in whom the insulin-producing β-cells of the pancreas were completely destroyed (and who only survived through the administration of insulin), the neighbouring α-cells in the islets of Langerhans of the pancreas (which normally secrete the antagonistic hormone glucagone) take over the production of insulin after a certain period of time. These results open up new perspectives for research into the causal therapy of type 1 diabetes.

In order to substantiate the finding that these new insulin-producing cells originated from α-cells, Herrera and his co-workers at the University of Geneva carried out a thorough analysis in which the “Tet-On Advanced System” was instrumental in determining that regenerated β-cells were derived from the α-cells in the islets of Langerhans. Boehnlein explained: “This publication is another powerful example of “recycling” previously established Tet mice when the generation of mice carrying multiple transgenes is required to address complex scientific questions.”

Publication: Thorel F, Népote V, Avril I, Kohno K, Desgraz R, Chera S, Herrera PL: Conversion of adult pancreatic α-cells to β-cells after extreme β-cell loss. Nature 464: 1149-1154 (2010).

Detailed information about this and many other applications of the Tet technology that have been published in leading scientific journals, is provided on the TET Systems website (see link in the above right-hand corner).

Further information:
Dr. Sabine Freundlieb
TET Systems GmbH & Co. KG
Im Neuenheimer Feld 582
69120 Heidelberg
Tel.: +49 (0)6221-58 804 00
Fax: +49 (0)6221-58 804 04
E-mail: info(at)tet-systems.com
Website address: https://www.gesundheitsindustrie-bw.de/en/article/news/tet-systems-controlled-gene-expression-in-eukaryotes