Tetracycline expression regulatory system

In 1992, Goseen et al. successfully constructed a tetracycline (Tet) eukaryotic cell gene regulatory expression system using prokaryotic gene regulatory elements. At present, this system has been widely applied in the fields of gene function and gene therapy research.

1. Basic principles of tetracycline regulated expression system

The Tet regulated expression system changes the conformation of regulatory proteins by inducing drugs such as tetracycline and doxycycline, thereby achieving the goal of regulating the expression of target proteins.

The Tet regulated gene expression system is based on the Tet resistance operon on the Tn10 transposon of Escherichia coli. The Tet repressor protein (TetR) can specifically bind to the Tet operator (TetO). When there is no inducing drug present, TetR binds to TetO, thereby blocking the expression of downstream resistance genes; When there is an inducing drug present, the conformation of TetR will change and detach from TetO, allowing downstream resistance genes to be expressed and bacteria to acquire resistance.

2.Tet on control system

By utilizing the specific binding characteristics of TetR and TetO, various types of Tet regulatory systems have gradually developed. Among them, the Tet on activated system is widely used.

The Tet on system consists of regulatory expression vectors and reactive expression vectors. The regulatory expression vector contains a human cytomegalovirus early promoter (PhCMV) and a reverse tethering transcriptional activator (rtTA). Among them, rtTA is formed by the fusion of reverse TetR (rTetR) and a transcriptional activation region at the C-terminus of herpes simplex virus (HSV) VP16 protein. The expression vector consists of Tet responsive element (TRE), minimal CMV promoter (PminCMV), and target gene. Among them, TRE consists of 7 repeated TetO sequences.

Tet on principle:

RTTA is formed by a mutation of 4 amino acids in tTA, and its phenotype is opposite to tTA. Under physiological conditions, rtTA does not bind to TRE, and due to the lack of enhancers in PminCMV, the target gene is not expressed; After being given DOX, the combination of DOX and rtTA binds to TRE, thereby initiating gene expression.

Tet off principle:

Tet transcriptional activator (tTA) is composed of 1-207 amino acids of TetR and 127 amino acids at the C-terminus of the activation domain of herpes simplex virus VP16. The increase of VP16 transforms TetR from a transcriptional repressor to a transcriptional activator. Under physiological conditions, tTA actively binds to TRE and initiates gene expression; If given DOX, the binding of DOX and tTA dissociates from TRE, thereby inhibiting gene expression.

3.Advantages of Tet on regulatory system

The Tet on regulatory system has four major characteristics:

high efficienc:The expression level of the target gene is low without induction, and the maximum induction fold can reach up to 10,000-fold.The TetR and TetO proteins of prokaryotic origin specifically bind to each other, and there are no analogous target sequences in mammals.

Precision:The drug dosage and administration time are controllable.

Safety:Tet and its derivatives have been used for many years and are considered safe and reliable. Good results can be achieved with low doses.

Reversibility:Removal of the inducer can shut down the system.
The inducer can be repeatedly added to initiate induction multiple times.

4.Weizhen Tet on/off service project

The Tet on expression regulatory system is currently the most widely used tetracycline regulatory system, so below, XiaoV will briefly introduce the service projects that Weizhen can provide using Tet on as an example.

Weizhen Biotechnology currently has multiple Tet on expression regulatory vectors, which can be packaged into teton adenovirus, lentivirus, and adeno-associated virus according to customer needs. Currently, Vizen is using third-generation technology (TRE3G). Compared to the previous two generations, the third generation technology has a lower background and higher drug sensitivity.