Tetracycline-Controlled Transcriptional Activation is a method of inducible expression where transcription is reversibly turned on or off in the presence of the antibiotic tetracycline or one of its derivatives (e.g. doxycycline). In nature, the Ptet promoter expresses TetR, the repressor, and TetA, the protein that pumps tetracycline antibiotic out of the cell.[1]
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The two most commonly used inducible expression systems for research of eukaryote cell biology are named Tet-Off and Tet-On.[2] They consist of a fusion of the Tet repressor and a VP16 activation domain to create a transcriptional activator protein (transactivator) rather than a repressor. Gene expression is activated as a result of binding of the Tet-Off or Tet-On protein to tetracycline response elements (TREs) located within an inducible promoter. The difference between Tet-On and Tet-Off is not whether the transactivator turns a gene on or off as the name might suggest; rather, both proteins activate expression. The difference relates to their respective response to doxycycline (Dox, a more stable tetracycline analogue); Tet-Off activates expression in the absence of Dox, whereas Tet-On activates in the presence of Dox.
The Tet-Off system for controlling expression of genes of interest in mammalian cells was developed by Professors Hermann Bujard and Manfred Gossen at the University of Heidelberg.[3] This system makes use of the tetracycline transactivator (tTA) protein created by fusing one protein, TetR(tetracycline repressor), found in Escherichia coli bacteria with another protein, VP16, produced by the Herpes Simplex Virus.[4] The tTA protein binds on DNA at a 'tet'O operator. Once bound the 'tet'O operator will activate a promoter coupled to the 'tet'O operator, activating the transcription of nearby gene. Tetracycline derivatives bind tTA and render it incapable of binding to TRE sequences, thereby preventing transactivation of target genes. This expression system is also used in generation of transgenic mice, which conditionally express gene of interest.
The Tet-On system works in the opposite fashion. In this system, the rtTA protein is capable of binding the operator only when bound by doxycycline. Thus the introduction of doxycyline to the system initiates the transcription of the genetic product. The Tet-On system is sometimes preferred for the faster responsiveness.
The Tet-On Advanced transactivator (also known as rtTA2S-M2) is an alternative version of Tet-On that shows reduced basal expression, and functions at a 10-fold lower Dox concentration than Tet-On. In addition, its expression is considered to be more stable in eukaryotic cells due to being human codon optimized and utilizing 3 minimal transcriptional activation domains. It was discovered in 2000 as one of two improved mutants by H. Bujard and his colleagues after random mutagenesis of the Tet Repressor part of the transactivator gene.[5] Tet-On 3G (also known as rtTA-V10) is similar to Tet-On Advanced because they were derived from the same predecessor. It is also human codon optimized and composed of 3 minimal VP16 activation domains. However, the Tet-On 3G protein has 5 amino acid differences compared to Tet-On Advanced which appear to increase its sensitivity to Dox even further. Tet-On 3G is sensitive to 100-fold less Dox than the original Tet-On.[6]
The tetracycline response element consists of 7 repeats of the 19bp bacterial tet-o sequence separated by spacer sequences. It is the tet-o that is recognized and bound by the TetR portion of Tet-On or Tet-Off. The TRE is usually placed upstream of a minimal promoter that has very low basal expression in the absence of bound Tet-Off (or Tet-On).
The Tet system has advantages over Cre, FRT, and ER (estrogen receptor) conditional gene expression systems. In the Cre and FRT systems, activation or knockout of the gene is irreversible once recombination is accomplished, whereas, in Tet and ER systems, it is reversible. The Tet system has very tight control on expression, whereas ER system is somewhat leaky. However, the Tet system, which depends on transcription and subsequent translation of a target gene, is not as fast-acting as the ER system, which stabilizes the already-expressed target protein upon hormone administration. Also, since the 19bp tet-o sequence is naturally absent from mammalian cells, pleiotropy is thought to be minimized compared to hormonal methods of control. When using the Tet system in cell culture, it is important to confirm that each batch of fetal bovine serum is tested to confirm that contaminating tetracyclines are absent or are too low to interfere with inducibility.