Genetically engineered mice are invaluable biological tools in biomedical research. The development of both transgenic and targeted mutant mice has allowed researchers to study gene function in the context of a whole mammalian organism. These studies, however, have limitations. For example, the deletion of a gene required during embyronic development often results in embryonic or perinatal lethality, making it impossible to study the effects of the gene ablation at later developmental ages.
In addition, unregulated over-expression of transgenic gene products may have unwanted physiological or toxic effects. The development of inducible expression systems has allowed researchers to overcome some of the problems associated with transgenic and targeted mutagenesis studies.
The Tet-Off and Tet-On expression systems are binary transgenic systems in which expression from a target transgene is dependent on the activity of an inducible transcriptional activator. In both the Tet-Off and Tet-On systems, expression of the transcriptional activator can be regulated both reversibly and quantitatively by exposing the transgenic animals to varying concentrations of tetracycline (Tc), or Tc derivatives such as doxycycline (Dox).
The Tet-Off and Tet-On systems are complementary, and the decision to choose one over the other depends on the particular experimental strategy. In the Tet-Off system, transcription is inactive in the presence of Tc or Dox; conversely, in the Tet-On system, transcription is active in the presence of Dox. (Tet-On responds poorly to Tc.)
Tet-Off expression system
In the Tet-Off expression system, a tetracycline-controlled transactivator protein (tTA), which is composed of the Tet repressor DNA binding protein (TetR) from the Tc resistance operon of Escherichia coli transposon Tn10 fused to the strong transactivating domain of VP16 from Herpes simplex virus, regulates expression of a target gene that is under transcriptional control of a tetracycline-responsive promoter element (TRE).
The TRE is made up of Tet operator (tetO) sequence concatemers fused to a minimal promoter, (commonly the minimal promoter sequence derived from the human cytomegalovirus (hCMV) immediate-early promoter). In the absence of Tc or Dox, tTA binds to the TRE and activates transcription of the target gene. In the presence of Tc or Dox, tTA cannot bind to the TRE, and expression from the target gene remains inactive.
Tet-On expression system
The Tet-On system is based on a reverse tetracycline-controlled transactivator, rtTA. Like tTA, rtTA is a fusion protein comprised of the TetR repressor and the VP16 transactivation domain; however, a four amino acid change in the tetR DNA binding moiety alters rtTA's binding characteristics such that it can only recognize the tetO sequences in the TRE of the target transgene in the presence of the Dox effector. Thus, in the Tet-On system, transcription of the TRE-regulated target gene is stimulated by rtTA only in the presence of Dox.
The two vector design of the Tet-Off and Tet On systems allows tissue-specific promoters to drive tTA or rtTA expression, resulting in tissue-specific expression of the TRE-regulated target transgene. Further, the ability to strictly regulate the level of rtTA and tTA activity allows the investigator to regulate activation of the target gene both quantitatively and temporally.
Important note: expression of a TRE-regulated transgene may be influenced also by its chromosomal insertion site.
References
1. Baron U, Bujard H. 2000. Tet repressor-based system for regulated gene expression in eukaryotic cells: Principles and advances. Methods Enzymol 327: 401-21.
2. Furth P et al. 1994. Temporal control of gene expression in transgenic mice by a tetracycline-responsive promoter.Proc Natl Acad Sci USA 91: 9302-6.
3. Gossen M, Bujard H. 1992. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 89: 5547-51.
4. Kistner A et al. 1996. Doxycycline-mediated quantitative and tissue-specific control of gene expression in transgenic mice. Proc Natl Acad Sci USA 93:10933-38.
5. Schonig, K, Bujard H. 2003. Generating conditional mouse mutants via tetracycline-controlled gene expression. In: Transgenic Mouse Methods and Protocols, Hofker, M, van Deursen, J (eds.) Humana Press, Totowa, New Jersey, pp. 69-104.