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Example of a T-REx system controlling the expression of shRNA. Tetracycline-controlled transcriptional activation is a method of inducible gene expression where transcription is reversibly turned on or off in the presence of the antibiotic tetracycline or one of its derivatives (e.g. doxycycline). [1]
The overall structure of TetR can be broken down into two DNA-binding domains (one per monomer) and a regulatory core, which is responsible for tetracycline recognition and dimerization. TetR dimerizes by making hydrophobic contacts within the regulatory core. There is a binding cavity for tetracycline in the outer helices of the regulatory domain.
To conduct an inducible lineage-tracing experiment two components must be engineered into the mouse genome: a switch and a reporter. The switch is commonly a drug-regulated form of the bacterial enzyme Cre-recombinase. This enzyme recognizes specific sequences, called LoxP sites. [63]
[1] [3] This whole system is inducible so a chemical can be added to knock genes out at a specific time. Two of the most commonly used chemicals are tetracycline, which activates transcription of the Cre recombinase gene and tamoxifen, which activates transport of the Cre recombinase protein to the nucleus. [4]
Members of the tetracycline class of antibiotics are often used as research reagents in in vitro and in vivo biomedical research experiments involving bacteria as well in experiments in eukaryotic cells and organisms with inducible protein expression systems using tetracycline-controlled transcriptional activation. [61]
Inducible systems - An inducible system is off unless there is the presence of some molecule (called an inducer) that allows for gene expression. The molecule is said to "induce expression". The manner by which this happens is dependent on the control mechanisms as well as differences between prokaryotic and eukaryotic cells.
Tetracycline hydrochloride is available as yellow crystalline powder. Since tetracycline is absorbed into bone, it is used as a marker of bone growth for biopsies in humans. Tetracycline labeling is used to determine the amount of bone growth within a certain period of time, usually a period around 21 days.
The promoters used in expression vector are normally inducible, meaning that protein synthesis is only initiated when required by the introduction of an inducer such as IPTG. Gene expression however may also be constitutive (i.e. protein is constantly expressed) in some expression vectors.