Review
Latest developments and in vivo use of the Tet system: ex vivo and in vivo delivery of tetracycline-regulated genes

https://doi.org/10.1016/S0958-1669(02)00361-0Get rights and content

Abstract

In June this year, the tetracycline-regulated gene expression system (tet system) celebrated its tenth ‘birthday’. In the past ten years a continuous stream of changes made to the tet system's basic components has led to a remarkable improvement in its overall performance. It was not until this year, however, that the full benefits of these improvements became apparent. In particular, usage of the tet system is no longer limited to immortalized cell lines and transgenic animals. In this review, we will describe the obstacles encountered in delivering the tet system's components to primary cells and tissues as well as the methods now used to overcome them. We will also focus on a novel system that is conceptually similar but based on different antibiotic/transcription factor pairs.

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)

  • U. Baron et al.

    Tetracycline-controlled transcription in eukaryotes: novel transactivators with graded transactivation potential

    Nucleic Acids Res

    (1997)
  • S. Urlinger et al.

    Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity

    Proc Natl Acad Sci USA

    (2000)
  • S. Lamartina et al.

    Stringent control of gene expression in vivo by using novel doxycycline-dependent trans-activators

    Hum Gene Ther

    (2002)
  • M. Fussenegger et al.

    Streptogramin-based gene regulation systems for mammalian cells

    Nat Biotechnol

    (2000)
  • C. Fux et al.

    Streptogramin- and tetracycline-responsive dual regulated expression of p27(Kip1) sense and antisense enables positive and negative growth control of Chinese hamster ovary cells

    Nucleic Acids Res

    (2001)
  • P. Tremblay et al.

    Doxycycline control of prion protein transgene expression modulates prion disease in mice

    Proc Natl Acad Sci USA

    (1998)
  • U. Deuschle et al.

    Tetracycline-reversible silencing of eukaryotic promoters

    Mol Cell Biol

    (1995)
  • F.M. Rossi et al.

    Tetracycline-regulatable factors with distinct dimerization domains allow reversible growth inhibition by p16

    Nat Genet

    (1998)
  • S. Freundlieb et al.

    A tetracycline controlled activation/repression system with increased potential for gene transfer into mammalian cells

    J Gene Med

    (1999)
  • Cited by (0)

    View full text