ReviewLatest developments and in vivo use of the Tet system: ex vivo and in vivo delivery of tetracycline-regulated genes
Section snippets
Introduction: the original tet system
The tetracycline-inducible system (or tet system) is comprised of three main components: the transcriptional modulator, the tetracycline-responsive promoter, and an antibiotic of the tetracycline family (Fig. 1). The transcriptional modulator consists of the prokaryotic tetracycline repressor (tetR), which provides tet-dependent DNA-binding activity, fused to a transcriptional activation (TA) or repression (TR or TS) domain that is active in eukaryotic cells. Both domains have been extensively
Past improvements
The original tetracycline-modulated transcription factor (tTA), obtained by fusing wild-type tetR with the herpes virus transcriptional activator VP16, still out-performs most later versions. Nevertheless, an important limitation of tTA is that it is inhibited, rather than activated, by tetracyclines (hence the alternative name, Tet-Off system). Subsequent mutations of the tetR moiety by Bujard and colleagues [2] resulted in the generation of a transcription factor with a reversed response to
Imitation is the most sincere form of flattery
The availability of a plethora of compounds already approved and routinely used in human therapy as well as the academic and commercial success of the tetracycline-inducible system have made antibiotic-regulated gene expression very attractive. Following the same concept underlying the tet system, Fussenegger's group [7••] developed a very similar regulation system from the pristinamycin-resistance operon of Streptomyces pristinaespiralis. All the components of the pristinamicin system are
New strategies for the generation of tet-inducible transgenics
The number of transgenic mouse lines expressing tet-system components in specific tissues continues to increase and several new ones have been described during the past year (Table 1). Nevertheless, the generation of these lines is traditionally a difficult task. Several factors are known to influence the efficiency of the tet system in transgenic animals, with the most important being the strain of mice and the site integration of the transgene [9•]. The generation of good tet-inducible lines
Delivering the tet system to primary cells — problems and solutions
In many cases for which inducible expression is desired in adult animals, a viable and potentially faster alternative to the production of transgenic lines is the direct delivery of the system's components to tissues or primary cells ex vivo. The same techniques form the basis of therapeutic gene delivery and have been the focus of much study in the past. This past year has demonstrated the integration of the tet system with delivery methods for primary cells that allow consistent, long-term
Conclusions
In summary, over the past year the most exciting news about tetracycline-inducible systems did not concern technological improvements to the system components themselves, but rather their successful use in several demanding and long-anticipated applications. In particular, many obstacles to delivering the tet system in vivo and ex vivo have been overcome. After ten years, the tet system has finally come of age.
Update
Fussenegger's group has recently reported the generation of yet another ‘tet-like system’ based on macrolides [38]. This system uses erythromycin or one of its homologues to modulate expression of the target gene. It is compatible with both the tetracycline- and the streptogramin-inducible systems, bringing to three the number of genes that can be independently regulated in the same cell. It is also available in both ‘OFF’ and ‘ON’ configurations.
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
References (38)
- et al.
Effects of mouse strain, position of integration and tetracycline analogue on the tetracycline conditional system in transgenic mice
Gene
(2002) - et al.
Use of the tetracycline-controlled transcriptional silencer (tTS) to eliminate transgene leak in inducible overexpression transgenic mice
J Biol Chem
(2001) - et al.
Robust and efficient regulation of transgene expression in vivo by improved tetracycline-dependent lentiviral vectors
Mol Ther
(2002) - et al.
Cell-type-specific and regulatable transgenesis in the adult brain: adenovirus-encoded combined transcriptional targeting and inducible transgene expression
Mol Ther
(2000) - et al.
Regulated expression of the human CFTR gene in epithelial cells
Mol Ther
(2001) - et al.
Recombinant AAV-mediated delivery of a tet-inducible reporter gene to the rat retina
Mol Ther
(2001) - et al.
Characteristics of adenovirus-mediated tetracycline-controllable expression system
Biochim Biophys Acta
(2001) - et al.
Tight control of gene expression in mammalian cells by tetracycline-responsive promoters
Proc Natl Acad Sci USA
(1992) - et al.
Transcriptional activation by tetracyclines in mammalian cells
Science
(1995) - et al.
Tet B or not tet B: advances in tetracycline-inducible gene expression
Proc Natl Acad Sci USA
(1999)