07-30-2013, 06:36 AM
We used cre-loxp technology to knockout one gene in mouse brain. We have confirmed the targeted gene was knockout at first two weeks after this gene deletion at protein and mRNA level. But I can not confirm it after 3 and 9 month of gene deletion.
Cre induced recombination is permanent and it cannot reverse later. But now how to explain our experiment? What are the possible reasons?
Recombinases are enzymes involved in site specific recombination, depending on the family and function these enzymes can be classified in to various categories.
Cre- Recombinases are a type of tyrosine recombinases found in P1 bacteriophages. Their basic mode of action is similar to that of an integrase. Theses enzymes scan and bind to recognition sites of 34 base pairs called loxP sites and induce recircularization of the phage DNA. A major advantage of Cre/lox recombination is that this mode of recombination doesn’t require any cofactors.
Cre-recombinase forms a tetramer with loxP sites bringing them close together for recombination using formation of Holliday structure.
Cre/lox recombination has been used to perform conditional gene knockout experiments. This is done basically by flanking the gene of interest between loxP sites creating a floxed gene. Then, Cre-recombinase is used to excise the target gene causing gene knockout.
Even though there are various methods to breed a knockout mouse, usually mice containing Cre-recombinase are bred separately and are then crossed with mice containing loxP sites.
Like floxed strains Cre expressing strains containing a transgene that expresses Cre under the control of a widespread (general) or tissue-specific (conditional) promoter. They have been used to produce general or conditional knockouts respectively.
Another mechanism of producing Cre/lox knockouts is using Cre reporter strains. These strains contain loxP sites along with a suitable selectable marker (fluorescent or lacZ) which makes identification of recombinants easier.
The last mechanism of producing Cre/lox recombinants is using inducible Cre strains which contain a transgene that expresses a modified form of Cre-recombinase that is inactive till an inducing agent like tamoxifen is added. The addition of inducing agent has to be done carefully as it requires the embryo or adult organism to be at as specific phase of the growth cycle.
In the experiment performed by you, the reversal of the recombination may be due to the disruption of the activity of the inducing agent. This has been seen in previous experiments involving tamoxifen.