诱导性基因表达系统 z-bo 2020-10-14 503

　　Inducible Gene Expression System (Inducible Gene Expression System) allows introduced genes to regulate gene expression levels at a specific time or on specific tissues or cell types. Currently, the most widely used inducible expression system is based on the inducible tetracycline operon regulatory system Tet-On/Tet-Off and the estrogen receptor and hormone phenoxyphene system.

　　(1) Tet controllable gene expression system: So far, the most widely used controllable gene expression system in transgenic mice is the tet gene expression control system based on the E. coli tetracycline (tet) operon. The control mechanism of the tet operon is realized by the tet suppressor (tetrepressor, tetR). The tetR protein binds to DNA called tetoperator (tetO) and performs transcriptional inhibition. Tetracycline prevents tetR from binding to tetO, thereby reversing the transcriptional suppression caused by tetR. By fusing the DNA binding domain sequence of the tetR gene with the gene expression activation domain sequence of simple herpes virus VP16, tetracycline-controlled transcription activator (tTA) can be expressed. tTA must be combined with an appropriate Tet-responsive promoter to function. Tet-responsive promoter is a series of tetO repeats (TRE) located upstream of the minimal promoter of cytomegalovirus (CMV), which can interact with VP16. The activation domains are properly aligned so that tTA binding can activate transcription.

　　(2) Tet-Off system: In the absence of tetracycline, the tTA protein continues to act on the tet promoter, allowing continuous gene expression. This system is very useful when it is necessary to maintain the introduced gene in a continuous expression state. By adding tetracycline, tetracycline can be combined with tTA to change the structure of tTA protein and prevent it from binding to the promoter, thereby reducing the level of gene expression it drives. In order to keep the system off, you need to continuously add tetracycline. Tet-On system: When studying mutant tTA genes, some variants (recombinanttTA, rtTA) no longer bind to TRE, and rtTA only binds to the TRE sequence in the presence of tetracycline to generate activation. It has been found to be capable of gene expression. It is called the Tet-On system because it is expressed by adding a tetracycline gene. When using this system. In the absence of tetracycline, no transgene is expressed. Only when tetracycline is added, rtTA will bind to TRE and induce transcription. In practice, it is usually necessary to establish two types of transgenic mice. One is the tTA (Tet-Off) or rtTA (Tet-On) transgenic mice expressed by tissue-specific promoters or other promoters, and the other is the transgenic mice expressing target genes. The antigene was cloned into the TRE-CMVmin promoter. After the child. After obtaining two transgenic mice, two transgenic mice were bred, and one mouse produced two transgenic mice at the same time. Later, it was found that the tetracycline analogue Doxcycline (Dox) is more effective. We are currently using doxcycline instead of tetracycline for experiments.

　　(3) Ligangen-induced expression gene regulation system: a commonly used estrogen receptor gene expression regulation system. Many studies have shown that the expression of protein activity can be regulated in a hormone-ligand dependent manner. For example, the ligand binding domain (LBD) of the steroid hormone receptor is fused to an inactive regulatory protein. The LBD binding of the ligand can change the composition of the fusion protein and cause protein activation. .. The most commonly used system is the estrogen receptor gene expression regulation system. The LBD sequence of the estrogen receptor is fused with a variety of different protein gene sequences to produce a fusion protein whose activity is regulated by the ligand. Such ligand-inducible proteins include neoplastic proteins, transcription factors, RNA binding proteins, and the like. The commonly used transgene regulatory system is the Cre-ER system described below.