Modifications and Refinements

Modifications and Refinements

Although the Tet System in its original version has been and still is in wide use, a number of modifications and refinements have significantly improved its applicability [3]. These modifications concern the tTA/rtTA responsive promoters, P_tet, 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. P_tet Derivatives
2. Novel Transactivators
3. Transcriptional Silencers
4. Viral and Episomal Vectors
5. Tet control in Whole Organisms
6. List of Tet Components

P_tet Derivatives

P_tet-bi is a bidirectional tTA/rtTA responsive promoter [4] where the heptamerized tetO sequences are flanked by two minimal promoters, thus allowing the coregulation of two genes transcribed in opposite directions (Fig. 2) . This promoter has proven particularly useful for monitoring gene activities that cannot be readily assayed. Coregulation of a target gene with a gene encoding an appropriate reporter function, thus, provides an assayable correlate. Likewise, P_tet-bi allows the control of the synthesis for two gene products that may form heterodimers. Finally, by fusing different minimal promoters to the tetO heptamer, two gene products may be synthesized at different but defined levels. P_tet-bi constructs are particularly useful in the generation of transgenic mouse lines, where for example the monitoring of Tet-dependent luciferase activities greatly facilitates the identification of founder lines with the expected regulation pattern.

P_tet-14 is a sequence variant of the original P_tet-1. Accordingly, P_tet-14 differs from P_tet-1 in the spacer sequence between the tetO elements which was shortened by 5 bp and redesigned to eliminate potential binding sites for endogenous mammalian transcription factors to the P_tet promoter region. Furthermore the CMV promoter part was reduced and now comprises CMV sequences from position -53 to +12 (Fig. 2) Remarkably, this promoter shows a significantly reduced background activity when used under transient expression conditions.

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 (Fig. 3) . This resulted in a set of transactivators that exhibit a graded activation potential depending on the number and sequence of the „acid domains“ [5] .
tTA2 corresponds in its activation potential 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 in improved yields and stability [6] .

rtTA2-syn series. Despite its wide and successful application, the original rtTA exhibits some limitations. It is fully activated only at relatively high Dox concentrations (1-2 µg/ml), which may be difficult to reach in some compartments of the mouse. Moreover, rtTA showed a low but distinct residual affinity to tetO, an issue in situations of transient expression and in episomal approaches.
Using genetic strategies, a series of novel rtTAs was identified, one of which exhibited highly 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(Fig. 3) (published in [6] as rtTA2^S-M2) exhibits a hardly measurable residual affinity to tetO and is fully induced at Dox concentrations as low as 80 ng/ml.

Transcriptional Silencers

tTS, tetracycline controlled transcriptional silencers are fusions between TetR and transcriptional silencing domains [7] . They were developed to shield the minimal promoter within P_tet from nearby transcriptional enhancers, which may cause a Tet-independent background activity often referred to as „leakiness“. tTS^Kid 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 heterodimerization when used in combination with rtTA, as shown in Fig. 4 . The tetracycline dependent repression-activation principle as outlined in Fig. 4 is particularly useful for tightly controlling P_tet-driven transcription units that cannot be sufficiently insulated from neighbouring enhancers. Such situations are frequently encountered with viral and particularly episomal vectors. tTS^Kid 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.

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, adenoviruses, adeno-associated viruses (AAV), Herpes simplex virus (HSV) and lentiviruses. These vectors allow fast and efficient transfer of the Tet System into tissue culture cells and animals. More importantly, regulated gene expression and efficient transfer are essential prerequisites for successful gene therapy. Here, inducibility of gene expression appears mandatory, not only for therapeutic fine tuning of the gene product to be delivered, but also for safety reasons.
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 cotransduction 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 P_tet may lead to interference, potentially resulting in a less stringent regulation.

As for viral vectors, crosstalk between the regulatory elements of the Tet System is 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, given appropriate design of the vectors, 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.

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.

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, providing new insights into such fundamental biological processes as development, behaviour and disease [8,9] .
For biomedical research, the ability to quantitatively and reversibly control disease genes has opened up new perspectives for modelling human diseases. Numerous disease models have, in the meantime, been described that permit the study not only of the onset of a disease, but also its progression, its potential reversibility and 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 an unique position as the standard for transcriptionally regulated transgenic mouse models, 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 around 100 mouse lines were published containing various target genes under P_tet control. Obviously, the combinatorial potential of this quickly expanding „zoo“ is only beginning to be exploited.

List of Tet components

All plasmids, viruses and cell lines that use Tet Technology are available through BD Biosciences Clontech (http://www.clontech.com/clontech/tet/index.shtml). 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: (http://www.clontech.com/clontech/products/literature/pdf/productlists/tetprodlist.pdf)

Nomenclature of plasmids generated by the Bujard laboratory that can be obtained from BD Biosciences Clontech

Tet System Components
	Tet Component / encoding Plasmid as published	Nomenclature of Tet Component by TET Systems	BD Clontech Plasmid Designation	Remarks
	tTA / pUHD 15-1neo [1]	tTA	pTet-Off	expression vector for tTA
	rtTA / pUHD 17-1neo [2]	rtTA	pTet-On	expression vector for rtTA
	tTA2 / pUHD 20-1 [3]	tTA2	ptTA2	expression vector for tTA with minimal domains
	tTA3 / pUHD 19-1 [3]	tTA3	ptTA3	expression vector for tTA with minimal domains
	tTA4 / pUHD 26-1 [3]	tTA4	ptTA4	expression vector for tTA with minimal domains
	tTA2^S / pUHT 61-1 [4]	tTA2-syn	will be available soon	expression vector for a synthetic, improved tTA
	rtTA2^S-M2 / pUHrT 62-1 [4]	rtTA2-syn1	will be available soon	expression vector for a synthetic, improved rtTA
	tTS Kid / pUHS 6-1 [5]	tTS ^Kid	pTet-tTS	expression vector for tTS

	P_hCMV*-1 / pUHD 10-3 [1]	P_tet-1	pTRE	cloning vector for genes of interest
	P_bi-1 / pBI-1 [6]	P_tet-bi	pBI-GL	bidirectional cloning vector controlling lacZ and luciferase
	P_bi-1 / pBI-3 [6]	P_tet-bi	pBI-G	bidirectional cloning vector controlling lacZ and gene of interest
	P_bi-1 / pBI-4 [6]	P_tet-bi	pBI	bidirectional cloning vector controlling two genes of interest
	P_bi-1 / pBI-5 [6]	P_tet-bi	pBI-L	bidirectional cloning vector controlling luciferase and gene of interest
	P_tet-14 / pUHC 13-3-1* [unpublished]	P_tet-14	pTRE-Tight	modified cloning vector for genes of interest

	P_hCMV*-1 / pUHC 13-3 [7]	P_tet-1	pTRE-luc	control vector for luciferase under tTA/rtTA control
	P_tetO-13 / pUHC 13-13 [5]	P_tetO-13	ptTS-Control	control vector for luciferase under tTS control

Antibodies
Polyclonal sera and monoclonal antibodies against TetR, which therefore recognize all of the transactivator variants listed above, are available from MoBiTec (http://www.mobitec.de/int/products/bio/05_antibodies/index.php?anti_tet.html).