Cre-Lox recombination

The Cre / loxP system is a system developed in the 1980s and patented by DuPont recombination system. Allows the selective removal of the DNA sequences in the living organisms. With this technique, for example, individual -specific cell or tissue types can be genetically modified, while other tissues remain unaffected. Originally, this system is derived from the bacteriophage P1 and will be held today esp. in molecular biology research and for the production of genetically modified plants used. An analog system, but is used less frequently, is the FLP - FRT system.

Operation

Cre ( cyclization recombination or Causes recombination ) or Flp (named after the flippase activity by the yeast sequence segments invert ) are enzymes of the class of recombinases. These proteins, which occur naturally in all organisms, catalyze the cleavage and rejoining of DNA between specific base sequences. This recognition sequence is referred to as loxP or FRT.

Is made ​​of a DNA strand in a specific DNA sequence or a specific gene to be removed, it is specifically in front of and behind this DNA section is a loxP site, which is referred to as floxen ( floxed = flanked by loxP, loxP sites of flanked ). The Cre enzyme recognizes and binds the respective loxP sites. This cuts out the corresponding DNA sequence or the gene when the two loxP sequences are oriented in the same direction ( excision). The two remaining loxP ends are joined together, the resulting short circular DNA portion is degraded in the cell. The two loxP sequences at the DNA oriented in the opposite direction, the enzyme catalyses the inversion (i.e., the swapping of the two ends ) of the floxed portion which remains is rotated by 180 ° in the DNA strand.

Applications

Plant

The Cre / loxP system is used for example, specifically to eliminate marker genes from transgenic plants. It is thus possible to produce transgenic organisms without then a selection on the basis of herbicide or antibiotic resistance, is required. This must first Cre recombinase temporarily ( transiently ) are transmitted into the cell. This transfer can be effected both tumefaciens using a plant virus ( PVX, TMV ) or by Agrobacterium. Triggered by the recombinase Cre recombination can be transferred to the next generation through regeneration or self-pollination. The progeny from self-pollination are when all reaction steps are successful, marker-free, but carry the gene of interest.

Animals

The system also works in mammalian cells reliably and without cofactors. Meanwhile, there is widely used for the production of tissue-specific knockout mice. In which tissue and when in an animal a gene is turned on, depends mainly on the associated promoter. Promoter and Cre can be introduced together as a transgene into the mouse genome. If, for example, genes are turned off only in the brain, one looks for a protein that is found only there and sets the associated promoter in front of the Cre gene, which Cre recombinase is formed only in the brain.

Now, a second mouse line is needed, in which the gene to be turned off is gefloxt. If the two mice now mated, we obtain, among other descendants who carry both genetic changes in their genetic material. A protein that is found in every cell in the body is, for example, no longer only formed in the brain, because the gene is made ​​there unusable by the recombinase.

A disadvantage of this is that most promoters will soon be activated in the embryonic development. If an important gene off early, the animals are often not viable and can not be examined. To work around this problem, methods have been invented that Cre can be turned on at any one time. This can be achieved eg by ligand - activated Cre recombinases. Cre was doing with the modified ligand binding domain (LBD ) fused by estrogen receptors, binds to the body's own estrogen no longer, but rather synthetic tamoxifen, an antiestrogen. The estrogen receptors are in the cytoplasm and are transferred only after binding of the ligand into the cell nucleus where the DNA is located. Thus, the associated with the estrogen receptor - Cre enzyme remains in the cytoplasm until tamoxifen is administered to the animals. As soon as it binds to the modified receptor, it is brought together with Cre in the nucleus, where Cre then cut out the floxed gene.