The tetracycline repressible system (tet-off) was the earliest system discovered to study the function of essential genes . It was developed at the University of Heidelberg in Germany by Manfred Gossen and Herman Boujard. They published their findings in the journal PNAS in 1992 where they used this system of conditional expression to study essential genes in mammalian cells. Later, Boujard and colleagues developed the tet-on or the tetracycline inducible system. It was first used in-vivo in mice and then later used in several other model organisms such as yeast, Dictyostelium, C.elegans, insects, zebrafish, Xenopus, rats, and non-human primates. (refernce link)
Tetracyclines and their derivatives are bacteriostatic drugs that were used to treat several diseases in humans and animals. They inhibit protein synthesis by preventing the tRNA from binding the A site of ribosomes during peptide elongation. A common drug used in this family is doxycycline. It easy diffuses into the body and can cross the blood brain barrier. The rise in tetracycline resistance in gram negative bacteria in the late 70s drove several researches to identify the mechanism behind it.
In 1980 a PNAS paper was published by Laura Mc Murry and colleagues from the Tufts school of Medicine , Boston that identified at least four different mechanism of tetracycline resistance. (PMID:7001450). They were all on plasmids and could be transferred to sensitive bacterial strains by conjugation and transfer resistance. The four plasmids were identified by DNA hybridization. It was observed that all four classes had some kind of an efflux mechanism to pump out the tetracycline and that this mechanism was associated to Tn10 transposon.
The tetR gene (tetracycline resistance regulatory gene) in Tn10 was identified in 1982 by Muller and colleagues at the University of Konstanz in West Germany. They did this using LacZ gene fusions and cloning. They found that there were at least two genes involved that have opposite orientations and that one gene product regulates the expression of the other. (PMID:6279565)
Further, the regulation of the transposon was identified by cloning the genes in Tn10 into vectors with lacZ fusions. (PMID:6327648). The two genes were identified in 1987-88 by restriction mapping and LacZ fusions and their sequences were obtained through Maxam and Gilbert Sequencing. (PMID:6330687). Figure two shows the map of the Tn10 transposon. The gene product of tetA was a metal-ion dependent transporter and it was transcribed by operator in the opposite orientation to the operator of the tetR gene which codes for a protein that acts as a repressor of the whole operon in its homodimeric conformation. The position and directionality of the two operators were identified by DNAseI footprinting. (PMID:2835235)
The crystal structure o
f the TetR binding the TetO operator was published in 2000 and it was shown that residues in the helix-turn-helix motif of the tetR are important for binding to the operator.( PMID:10700280)
This system was later modified and incorporated in 1992 by the German Scientists, Manfred Gossen and Herman Boujard. This conditional expression system is now used in several organisms from prokaryotes to Eukaryotes and has revolutionized the study of the effects of deleterious mutantions and the study of the function of essential genes.
The Inner Workings of the tet system
This system has three parts:
1. Tetracycline controlled trans-activator (tTA) : The Tet repressor gene(tetR) from the tetracycline resistance operon (Tn10) in is fused to activation domain of VP16 from Herpes simplex virus. This fusion protein is constitutively expressed in cells.
2. Minimal Promoter of cytomegalovirus(CMV) and E.coli derivedykywff tTA operator(tetO) upstream of gene of interest
3. Tetracyline family of drugs- Doxycycline and anhydrotetracycline are commonly used.
How does it work?
tetR is negatively regulated by tetracycline. When tTA is constitutively expressed in cells, it binds the tetO operator and a Certain mutations in tTA were later identified which required the physical binding of tetracycline derivatives for gene activation. This was named reverse tTA or rtTA. In this way, the gene of interest (mutant or wild-type) can be expressed upon addition of tetracycline. Figure 2 illustrates this system.
There have been several versions of this system with modifications that enables scientists to use this in several cell lines. It is better than the other systems to study the functions of essential genes or deleterious mutations as this effect is reversible. The level of expression and the timing depends on the presence or absence of tetracycline derivatives. Tetracycline derivatives are not harmful to cells and animals. The expression system is not leaky compared to other systems. This system has been successfully used to study several genes in transgeneic mice as well as other model organisms. There were a few modifications made over time in tetR sequence as well as structure based design of the tetR's. The codon usage adaptation was modified to express successfully in prokaryotes as well as eukaryotes. This system has been successfully used in Mycobaterium to study the function of important virulence genes which was otherwise very hard. (PMID:19174563)
Uses in Human Gene therapy: Merits and Concerns
The Tet- on system is an excellent way of delivering genes into tissues for gene therapy. There are still several obstacles to cross for its use in humans but the system is promising. The dose of doxycyline was too high for expression in the tet-on system but two mutants of rtTA were isolated which had minimal basal activity. The expression in the presence of doxycycline was made more stringent by using the dox- dependent trans-silencer called tTS. Here, a KRAB (Krupel Associated Box) of human kid-1 protein is fused to wt TetR. In the absence of doxycycline, tTS binds DNA and inhibits transcription . The effects are reversed in the presence of doxycycline. Fig3 illustrates this.
The tet system has been successful in mice as it is not immunogeneic in mice. There is a hesitation to use this in humnas for fear of an immune response to the VP16 from the Herpes simplex virus which may cause an adverse immune responnse and latent infection. It cant cross react with other herpes visruses as there is not enough sequence homology. For this reason the VP16 gene was replaced by a human gene p65 from the NF kappa beta pathway. It has been susscessful in brain tissue when delivered using lentiviral vectors.(PMID:22426391)
4. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3974-7. Active efflux of tetracycline encoded by four genetically different tetracycline resistance determinants in Escherichia coli.McMurry L, Petrucci RE Jr, Levy SB.
6. J Bacteriol. 1984 Jun;158(3):910-9. Construction of a single-copy promoter vector and its use in analysis of regulation of the transposon Tn10 tetracycline resistance determinant.Bertrand KP, Postle K, Wray LV Jr, Reznikoff WS.
11.Gene therapy progress and prospects: transcription regulatory systems. Gene Therapy (2004) 11, 649–657. doi:10.1038/sj.gt.3302251 Published online 26 February 2. C Toniatti1, H Bujard2, R Cortese1 and G Ciliberto1
[http://www.ncbi.nlm.nih.gov/pubmed?term=Gene%20therapy%20progress%20and%20prospects%3A%20transcription%20regulatory%20systems# ] [http://www.ncbi.nlm.nih.gov/pubmed?term=Gene%20therapy%20progress%20and%20prospects%3A%20transcription%20regulatory%20systems# ]