点特异性重组系统 z-bo 2020-10-14 314

　　"Point-specific recombination systems" include Cre-LoxP systems and FLP-FRT systems. So far, the most widely used method is the Cre/LoxP-mediated point-specific recombination system.

　　The Cre-LoxP system is most commonly used for conditional gene knockout and also for conditional expression of transgenes in vivo, including certain types of cells and certain developing tissues. The expression of the transgene can be achieved in a specific time and space. Expressed as process or adulthood. The Cre/LoxP point-specific recombination discovered in the 1980s has been successfully used in yeast, plants, cultured mammalian cells and mice. Cre (Causes Recombination) is a 38 kDa recombinase isolated from bacteriophage P1 that recognizes LoxP sites. The LoxP site is directional, because the LoxP [crossover point (x) located on P1] element is a 34bp long sequence composed of an ileum sequence with 13bp at both ends and an asymmetric sequence with 8bp at the center. Cre recombinase can mediate the recombination reaction between two LoxP elements, each repeat sequence can be combined with Cre molecules, and recombination occurs in the spacer region. Two LoxP elements can be placed on the same or different DNA strands. If two LoxP elements on the same DNA strand have the same direction, the Cre enzyme will mediate the excision and circularization of the DNA sequence between LoxP, while only one LoxP element remains in the original sequence, while in the other LoxP Keep one LoxP element. The site sequence enters the loop by recombination.

　　The "circularized DNA sequence" lacks DNA replication sequences and centromeres, and is lost as the cell divides. When the directions of the two elements are different, the Cre enzyme mediates the restoration of the DNA sequence between the LoxP elements, resulting in a change in direction. When two LoxP elements are located on different DNA strands, Cre enzyme mediates recombination between strands or transfer between different DNA strands, exchanging large fragments. The recombination reaction by the Cre enzyme is also bidirectional. For example, cut circular DNA can also recombine back to the DNA strand, but for kinetic reasons, Cre enzyme recombination tends to be cut rather than inserted. Cre-LoxP can be used to control the expression of transgenes. First, insert a stop sequence (LoxP-Stop-LoxP) (stop sequence or Stop box) with two LoxP sites before and after the promoter and the controlled transgene. The termination cassette translates the end expression sequence to prevent the expression of the transgene. With Cre enzyme activity, the expression termination sequence between LoxP is deleted, so that the transgene can be translated and expressed, thus opening the silent transgene. Using cell-type-specific promoters to control Cre expression can activate Cre recombinase at specific developmental stages or under specific physiological conditions. Many tissue-specific Cre mice have been constructed and can be found in the Cre mouse library. For example, in the nervous system, the nestin gene (nesin) expressed in neuroepithelial cells (including glial cells) and neural progenitor cells is widely used to generate knockout mice throughout the nervous system. The second intron of the estin gene has two enhancers, the brain-specific enhancer and the pan-central nervous system enhancer, which can specifically express Cre in central nerve cells.

　　Carmodulin kinase dicalcium promoter is usually used to express Cre in brain neurons (including cerebral cortex and hippocampus) and cause gene inactivation in the brain. Saccharomyces cerevisiae has a similar point-specific recombination system Flp/FRT for generating inducible transgenic models. Flp Flipper recognizes the 34bp flippase recognition target sequence (flipperse recognition target, FRT). Flp and Cre can be used in combination. For example, Flp/FRT is used to remove selectable marker genes at the ES cell stage, while Cre is used to generate gene knockouts in vivo. Cre-ER system: The estrogen receptor (ER) variant has lost its ability to bind to endogenous estrogen, but it can still bind to the estrogen antagonist tamoxifen. The fusion of Cre and the estrogen receptor mutant gene constitutes the ER-Cre fusion protein; without induction, the fusion protein Cre dissociates in the cytoplasm and forms a complex with the inactive heat shock protein (hsp90). I will. The binding of the ligand tamoxifen disrupted the interaction between ER-Cre and hsp90, transported Cre to the nucleus, and used LoxP sites on both sides to excise the target DNA target sequence. The tamoxyphene system is known for its strict gene regulation, but gene resection is not effective in all tissues and organs. Cre recombinase-mediated gene regulation induces gene expression in mouse gene regulation and is a very sensitive system. However, Cre recombinase-mediated gene excision is one-time and irreversible.

　　Tool mouth: Use different universal or tissue-specific promoters and different inducible gene expression systems (Cre-LoxP or Tet-On/Tet-Off) and other key research proteins, such as EGFP and Cre/FLP recombinase Many important transgenic tools have been established in mice. The Jackson Institute and other institutions have collected these mice and established mouse banks for EGFP, Cre, fluorescein and Tet tools.