Cre / loxP system
The Cre / loxP system is a recombination system. It enables the targeted removal of DNA sequences in 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 comes from the bacteriophage P1 and is found today v. a. in molecular biological research and for the production of genetically modified plants. An analogous system is the Flp-FRT system.
principle
Cre (from the English cyclization recombination or causes recombination ) or Flp (named after the flippase activity by which yeast invert sequence segments) are enzymes of the recombinase class . These proteins , which are naturally found in all organisms, catalyze the cleavage and reconnection of DNA between specific base sequences . This recognition sequence is referred to as loxP or FRT . The Cre / loxP system was developed in the 1980s and patented by DuPont .
If a certain DNA sequence or a certain gene is to be removed from a DNA strand, a loxP position is placed in front of and behind this DNA section , which is referred to as floxing ( floxed = flanked by loxP ; dt loxP sites flanked '). The Cre enzyme recognizes and binds the respective loxP sites. It cuts out the corresponding DNA sequence or the gene if the two loxP sequences are oriented in the same direction ( excision ). The two remaining loxP ends are joined, the resulting short circular DNA segment is broken down in the cell . If the two loxP sequences on the DNA are oriented in opposite directions, the enzyme catalyzes the inversion (i.e. the exchange of the two ends) of the floxed section, which remains in the DNA strand rotated by 180 °.
Cre recombinase
The Cre recombinase (from English causes recombination or cyclization recombinase ) consists of four subunits with 343 amino acids each in two protein domains . The C-terminal domain of Cre recombinase contains the active center and is structurally similar to the integrase family from bacteriophage λ .
loxP
The loxP - DNA sequence (from English locus of X-over P1 ) comes from the bacteriophage P1 and consists of 34 base pairs , including an asymmetrical sequence of eight base pairs (the two in the middle of which are conserved ) flanked on both sides by palindromic thirteen bases are.
| 13bp | 8bp | 13bp |
| ATAACTTCGTATA - | NNNTANNN | -TATACGAAGTTAT |
| Surname | 13bp detection region | 8bp spacer region | 13bp detection region |
|---|---|---|---|
| Lox 511 | ATAACTTCGTATA | ATGTATaC | TATACGAAGTTAT |
| Lox 5171 | ATAACTTCGTATA | ATGTgTaC | TATACGAAGTTAT |
| Lox 2272 | ATAACTTCGTATA | AaGTATcC | TATACGAAGTTAT |
| M2 | ATAACTTCGTATA | AgaaAcca | TATACGAAGTTAT |
| M3 | ATAACTTCGTATA | taaTACCA | TATACGAAGTTAT |
| M7 | ATAACTTCGTATA | AgaTAGAA | TATACGAAGTTAT |
| M11 | ATAACTTCGTATA | aGATAgaa | TATACGAAGTTAT |
| Lox71 | taccgTTCGTATA | NNNTANNN | TATACGAAGTTAT |
| Lox66 | ATAACTTCGTATA | NNNTANNN | TATACGAAcggta |
Applications
plants
The Cre / loxP system is used, for example, to specifically eliminate marker genes from transgenic plants. In this way it is possible to generate transgenic organisms without having to subsequently select on the basis of herbicide or antibiotic resistance. To do this, Cre recombinase must first be temporarily (transiently) transferred into the cell. This transmission can take place both with the help of a plant virus ( PVX , TMV ) and by Agrobacterium tumefaciens . The recombination events triggered by the Cre recombinase can be transferred to the next generation via regeneration or self-pollination. The offspring from self-pollination are marker-free if all reaction steps have been successful, but they carry the gene of interest.
Animals
The system also works reliably in mammalian cells and without further cofactors . It is now widely used for the production of tissue-specific knockout mice . In which tissue and at which point in time in a living being a gene is switched on depends mainly on the associated promoter . Promoter and Cre are introduced together as a transgene into the mouse genome. Should z. If, for example, genes are only switched off in the brain, one looks for a protein that only occurs there and places the associated promoter in front of the Cre gene, whereby Cre recombinase is only formed in the brain.
Now a second mouse line is needed, where the gene that is to be switched off is flown. If the two mice are now paired, one obtains, among other things, offspring that both carry genetic changes in their genome. A protein that is found in every cell in the body is no longer produced in the brain, for example, since the gene there is rendered unusable by the recombinase.
A disadvantage here is that most promoters are activated very soon during embryonic development . If an important gene is switched off early on, the animals are often not viable and cannot be examined. To get around this problem, methods were invented that Cre can be switched on at any point in time. This succeeds z. B. with ligand-activatable Cre recombinases. Cre was fused with the modified ligand binding domain (LBD) of estrogen receptors to which the body's own estrogen no longer binds, but synthetic tamoxifen , an antiestrogen , does . The estrogen receptors are located in the cytoplasm and are only transferred to the cell nucleus , where the DNA is located, after the ligand has been bound . Thus, the Cre enzyme associated with the estrogen receptor also remains in the cytoplasm until tamoxifen is administered to the animals. As soon as this binds to the modified receptor, it is brought into the cell nucleus together with Cre, where Cre can then cut out the floxed genes.
See also
literature
- D. Metzger, P. Chambon : Site- and time-specific gene targeting in the mouse . In: Methods , 24 (1), 2001, pp. 71-80, PMID 11327805 .
- R. Feil, J. Wagner, D. Metzger, P. Chambon: Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains . In: Biochem Biophys Res Commun , 237 (3), 1997, pp. 752-757, PMID 9299439 .
Web links
Individual evidence
- ↑ Cre recombinase in GenBank.
- ↑ N. Sternberg, D. Hamilton: Bacteriophage P1 site-specific recombination. I. Recombination between loxP sites. In: Journal of molecular biology. Volume 150, Number 4, August 1981, pp. 467-486, ISSN 0022-2836 . PMID 6276557 .
- ↑ PI Missirlis, DE Smailus, RA Holt: A high-throughput screen identifying sequence and promiscuity characteristics of the loxP spacer region in Cre-mediated recombination. In: BMC genomics. Volume 7, 2006, p. 73, ISSN 1471-2164 . doi : 10.1186 / 1471-2164-7-73 . PMID 16595017 . PMC 1479339 (free full text).