Tetracycline controlled transcriptional activation
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 (etc. doxycycline). In nature, pTet promotes TetR, the repressor, and TetA, the protein that pumps tetracycline antibiotic out of the cell.[1] Two systems named Tet-off and Tet-on are used.
Tet-off
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[1] 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.[2] 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, therefore preventing transactivation of target genes. This expression system is also used in generation of transgenic mice, which conditionally express gene of interest.
Tet-on
The Tet-on system works in the opposite fashion. In that system the rtTA protein is only capable of binding the operator 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.
Tet system has advantages over Cre, FRT and ER (estrogen receptor) conditional gene expression systems. In Cre and FRT systems, activation of knockout of the gene is irreversible once recombination is accomplished, while in Tet and ER systems it is reversible. Tet system has very tight control on expression, while ER system is somewhat leaky. However, Tet system, which depends on transcription and subsequent translation of target gene, is not as fast acting as ER system, which stabilizes the already expressed target protein upon hormone administration.
See also
External links
- http://www.zmbh.uni-heidelberg.de/Bujard/default.shtml Hermann Bujard Lab, ZMBH, Heidelberg, Germany
- http://jaxmice.jax.org/models/tet_intro.html Jackson Laboratory
Notes
- ↑ Bujard, Hermann (1992). "Tight Control of Gene Expression in Mammalian Cells by Tetracycline-Responsive Promoters". Proceedings of the National Academy of Sciences. 89: 5547–5551. Unknown parameter
|coauthors=ignored (help) - ↑ Allen, Nicholas D. (2000). "Directed Mutagenesis in Embryonic Stem Cells". Mouse Genetics and Transgenics: 259–263. Unknown parameter
|coauthors=ignored (help)
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