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Modifications and Refinements
Although the Tet System in its original version has been and still is widely used, a number of modifications and refinements have significantly improved its utility . These modifications include improvements of the tTA/rtTA responsive promoters, Ptet, as well as the transcription activators tTA and rtTA. In addition, a new class of components has been developed: the tetracycline controlled transcriptional silencers, tTS.
1. Ptet Derivatives
Ptet-bi is a bidirectional tTA/rtTA responsive promoter  in which heptamerized tetO sequences are flanked by two minimal promoters. This allows the coregulation of two genes transcribed in opposite directions (Figure 2). This promoter has proven particularly useful for monitoring gene activities that cannot be assayed easily. Coregulation of a target gene with a gene encoding an appropriate reporter function provides a direct, assayable correlate. Furthermore, Ptet-bi allows the control of the synthesis of two gene products that may form heterodimers. Finally, by fusing different minimal promoters to the tetO heptamer, two gene products may be synthesized at defined but different levels. Ptet-bi constructs are particularly useful in the generation of transgenic mouse lines, where the monitoring of Tet-dependent luciferase activities greatly facilitates the identification of founder lines with the expected Tet-controlled regulation pattern.
Ptet-14 is a sequence variant of the original Ptet-1. Accordingly, Ptet-14 differs from Ptet-1 in the spacer sequence between the tetO elements. This element was shortened by 5 bp and redesigned to eliminate potential binding sites for endogenous mammalian transcription factors to the Pter region. Furthermore, the CMV promoter part was minimized and only comprises CMV sequences from position -53 to +12 (Figure 2). Remarkably, this promoter shows a significantly reduced background activity when used under transient expression conditions.
2. Novel Transactivators
tTA2-syn series. For a number of reasons, it appeared advantageous to replace the original VP16 transcription activation domain of 127 amino acids by so-called "acid domains" of only 13 amino acids (Figure 3). This resulted in a set of transactivators that exhibit a graded activation potential depending on the number and sequence of the "acid domains" .
In its activation potential, tTA2 compares to the original tTA. The coding sequence of tTA2 was redesigned reconciling a number of parameters for optimal expression in higher eukaryotes. The resulting tTA2-syn transactivator is produced in HeLa cells at higher yields and better stability .
rtTA2-syn series. Despite its wide and successful application, the original rtTA has some limitations. It is fully activated only at relatively high Dox concentrations (1-2 µg/ml), which may be difficult to reach in certain organs/tissues of the mouse. Moreover, rtTA showed a low but distinct residual affinity to tetO, which is undesirablein particular in transient expression studies and using episomal approaches.
Using genetic strategies, a series of novel rtTAs was identified. One of these series exhibited the desired, improved properties. The respective mutant TetR-M2 was fused to three minimal activation domains and the coding sequence was redesigned following the rules applied to the tTA2-syn gene. The resulting rtTA2-syn1 (Figure 3) (published in  as rtTA2S-M2) exhibits a hardly detectable residual affinity to tetO and is fully induced at Dox concentrations as low as 80 ng/ml.
3. Transcriptional Silencers
tTS, tetracycline controlled transcriptional silencers are fusions between TetR and transcriptional silencing domains . They were developed to shield the minimal promoter within Ptet from nearby transcriptional enhancers, which may cause a Tet-independent background activity often referred to as "leakiness". tTSKid is a fusion between TetR and the 62 amino acid KRAB silencing domain of the human gene kid-1. It has a modified TetR domain which prevents hetero-dimerization when used in combination with rtTA, as shown in Figure 4. The tetracycline-dependent repression-activation principle as outlined in Figure 4 is particularly useful for tightly controlled Ptet-driven transcription units that otherwise cannot be sufficiently insulated from neighboring enhancers. Such situations are frequently encountered with viral and particularly episomal vectors. tTSKid or similar TetR-based silencer proteins may also be used for the doxycycline-dependent control of other promoter/tetO combinations recognized by RNA polymerase II, III or I.
4. Viral and Episomal Vectors
To combine inducible expression with virally mediated gene transfer, the Tet Technology was integrated into a variety of viral vectors derived from retroviruses including lentiviruses, adenoviruses, adeno-associated viruses (AAV), and Herpes simplex virus (HSV). These vectors allow fast and efficient transfer of the Tet System into tissue culture cells and animals. More importantly, regulated gene expression and efficient gene transfer are also prerequisite for successful gene therapy. Here, inducibility of gene expression appears mandatory, not only for fine tuning of the therapeutic gene product to be delivered, but also for safety reasons to achieve a "therapeutic window".
As in the plasmid-borne system, approaches used for development of viral vectors can be divided into "one-virus" and "two-virus" strategies. When one considers potential problems of co-transduction, it appears advantageous to incorporate the transactivator gene and the response unit in a single vector. On the other hand, close proximity of the promoter driving transactivator expression and Ptet may lead to interference, potentially resulting in a less stringent gene regulation.
Crosstalk between the regulatory elements of the Tet System is also a concern whenever the components of the system are incorporated into a single episomal vector. The strategies to overcome such potential limitations are the same as discussed for viral vectors. However, it has been shown that, using an appropriate vector design, such systems can function efficiently even without the incorporation of insulator sequences or silencer proteins. Such vectors, especially those based on the Epstein-Barr-Virus (EBV) replicon have considerable potential for speeding up the establishment of the Tet System in cultured cells, whenever chromosomal integration of the regulatory system is not required.
5. Tet control in Whole Organisms
The general applicability of Tet regulation is most impressively demonstrated by the broad spectrum of transgenic organisms into which Tet control has been transferred. They include:
- unicellular organisms such as S. cerevisiae, Dictiostelium, Apicomplexa like Toxoplasma gondii and Plasmodium falciparum;
- insects such as Drosophila melanogaster and Anopheles gambiae;
- plants such as Arabidopsis, tobacco and moss;
- zebrafish (Danio rerio);
- amphibians such as Xenopus laevis;
- mammals, particularly mice and rats.
In addition, Tet regulation has been successfully transferred to mice, rats and non-human primates via viral vectors, naked DNA or cell transplants.
The most remarkable results have been obtained in transgenic mice, where the potential to control individual gene activities in a temporally defined and cell type-restricted manner has allowed the in vivo dissection of gene functions and pathways with unprecedented precision. These elegant studies provided new insights into such fundamental biological processes as development, behaviour and disease [8,9] or biomedical research. The ability to quantitatively and reversibly control disease genes has opened up new perspectives for modelling human diseases.Numerous disease models have been described that permit the study not only of the onset of a disease, but also its progression, its potential reversibility and disease regression. Needless to say, such models will more faithfully mimic pathologies and will thus enhance our understanding of diseases at the molecular and physiological level, thereby facilitating the development of new strategies for intervention and prevention.
The Tet Technology holds the unique position as the standard for transcriptionally regulated transgenic mouse models. It is unchallenged by any of the alternative technical approaches developed in the past decades for inducible regulation in tissue culture. More than 80 mouse lines have been described expressing the tTA/rtTA genes under the control of a variety of tissue specific promoters, and approximately 100 mouse lines have been published containing various target genes under Ptet control. Obviously, the combinatorial potential of this quickly expanding "zoo" is only beginning to be exploited.
6. List of Tet components
Clontech is the exclusive partner to market all plasmids, viruses and cell lines that use the Tet Technology. The following table lists only those reagents that were originally transferred from the Bujard laboratory to Clontech. For a complete listing of all available Tet reagents and, in particular, the wide range of Tet System responsive cell lines available, please visit: www.clontech.com
Nomenclature of plasmids generated by the Bujard laboratory that can be obtained from Clontech:
Tet System Components
|Tet Component / encoding Plasmid
|Nomenclature of Tet Component
by TET Systems
|BD Clontech Plasmid Designation||Remarks|
|tTA / pUHD 15-1neo ||tTA||pTet-Off||expression vector for tTA|
|rtTA / pUHD 17-1neo ||rtTA||pTet-On||expression vector for rtTA|
|tTA2 / pUHD 20-1 ||tTA2||ptTA2||expression vector for tTA with minimal domains|
|tTA3 / pUHD 19-1 ||tTA3||ptTA3||expression vector for tTA with minimal domains|
|tTA4 / pUHD 26-1 ||tTA4||ptTA4||expression vector for tTA with minimal domains|
|tTA2S / pUHT 61-1 ||tTA2-syn||will be available soon||expression vector for a synthetic, improved tTA|
|rtTA2S-M2 / pUHrT 62-1 ||rtTA2-syn1||will be available soon||expression vector for a synthetic, improved rtTA|
|tTS Kid / pUHS 6-1 ||tTS Kid||pTet-tTS||expression vector for tTS|
|PhCMV*-1 / pUHD 10-3 ||Ptet-1||pTRE||cloning vector for genes of interest|
|Pbi-1 / pBI-1 ||Ptet-bi||pBI-GL||bidirectional cloning vector controlling lacZ and luciferase|
|Pbi-1 / pBI-3 ||Ptet-bi||pBI-G||bidirectional cloning vector controlling lacZ and gene of interest|
|Pbi-1 / pBI-4 ||Ptet-bi||pBI||bidirectional cloning vector controlling two genes of interest|
|Pbi-1 / pBI-5 ||Ptet-bi||pBI-L||bidirectional cloning vector controlling luciferase and gene of interest|
|Ptet-14 / pUHC 13-3-1* [unpublished]||Ptet-14||pTRE-Tight||modified cloning vector for genes of interest|
|PhCMV*-1 / pUHC 13-3 ||Ptet-1||pTRE-luc||control vector for luciferase under tTA/rtTA control|
|PtetO-13 / pUHC 13-13 ||PtetO-13||ptTS-Control||control vector for luciferase under tTS control|