Cre-Lox recombination
Cre-Lox recombination is a site-specific recombinase technology, used to carry out deletions, insertions, translocations and inversions at specific sites in the DNA of cells.
The Cre-lox recombination system has been particularly useful to help neuroscientists to study the brain in which complex cell types and neural circuits come together to generate cognition and behaviors.
Removal of selectable markers from the genome by Cre-lox recombination is an elegant and efficient way to circumvent this problem and is therefore widely used in plants, mouse cell lines, yeast, etc.
The activity of the Cre enzyme can be controlled so that it is expressed in a particular cell type or triggered by an external stimulus like a chemical signal or a heat shock.
[3][4][5] Subsequently, researchers in the laboratory of Dr. Jamey Marth demonstrated that Cre-Lox recombination could be used to delete loxP-flanked chromosomal DNA sequences at high efficiency in specific developing T-cells of transgenic animals, with the authors proposing that this approach could be used to define endogenous gene function in specific cell types, indelibly mark progenitors in cell fate determination studies, induce specific chromosomal rearrangements for biological and disease modeling, and determine the roles of early genetic lesions in disease (and phenotype) maintenance.
[6] Shortly thereafter, researchers in the laboratory of Prof. Klaus Rajewsky reported the production of pluripotent embryonic stem cells bearing a targeted loxP-flanked (floxed) DNA polymerase gene.
Researchers have since reported more efficient Cre-Lox conditional gene mutagenesis in the developing T cells by the Marth laboratory in 1995.
[9] Incomplete deletion by Cre recombinase is not uncommon in cells when two copies of floxed sequences exist, and allows the formation and study of chimeric tissues.
[10] Tsien and his colleagues demonstrated Cre-mediated recombination can occur in the post-mitotic pyramidal neurons in the adult mouse forebrain.
Cre-Lox recombination involves the targeting of a specific sequence of DNA and splicing it with the help of an enzyme called Cre recombinase.
Cre-Lox recombination is commonly used to circumvent embryonic lethality caused by systemic inactivation of many genes.
[15][16] As of February 2019, Cre–Lox recombination is a powerful tool and is used in transgenic animal modeling to link genotypes to phenotypes.
[12][17][18] The Cre-lox system is used as a genetic tool to control site specific recombination events in genomic DNA.
If loxP sites are on different chromosomes it is possible for translocation events to be catalysed by Cre induced recombination.
An additional level of control can be achieved by using his Cre recombinase, engineered to reversibly activate in the presence of the estrogen analogue 4-hydroxy tamoxifen.
This tool is suitable for deleting antibiotic resistance genes, but above all it allows conditional knockouts that can be induced at specific times in the cell type of choice.
This model provided convenient explanation for the strict requirement for homology between recombining sites, since branch migration would stall at a mismatch and would not allow the second strand exchange to occur.
In more recent years, however, this view has been challenged, and most of the current models for Int, Xer, and Flp recombination involve only limited branch migration (1–3 base pairs of the Holliday intermediate), coupled to an isomerisation event that is responsible for switching the strand cleavage specificity.
Site-specific recombination (SSR) involves specific sites for the catalyzing action of special enzymes called recombinases.
Site-specific recombination is also an important process that viruses, such as bacteriophages, adopt to integrate their genetic material into the infected host.
Increasing the length of DNA leads to decreased efficiency of Cre/lox recombination possibly through regulating the dynamics of the reaction.
[27][28][29] Genetic location of the floxed sequence affects recombination efficiency as well probably by influencing the availability of DNA by Cre recombinase.
[29] Failure to activate both recombination events simultaneously confounds the interpretation of cell fate mapping results.
Once tamoxifen is introduced, it is metabolized into 4-hydroxytamoxifen, which then binds to the ER and results in the translocation of the CreER into the nucleus, where it is then able to cleave the lox sites.
Therefore, researchers often use transgenic mice expressing CreERt2 recombinase induced by tamoxifen administration, under the control of a promoter of a gene that marks the specific cell type of interest, with a Cre-dependent fluorescent protein reporter.
CreER(T2) resides within the cytoplasm and can only translocate to the nucleus following tamoxifen administration, allowing tight temporal control of recombination.
Since removal of the stop cassette is permanent, the reporter genes are expressed in all the progeny produced by the initial cells where the Cre was once activated.
It is also proposed that rolling circle replication followed by recombination will allow the plasmid to increase its copy number when certain regulators (repA) are limiting.
[39] A classical strategy for generating gene deletion variants is based on double cross-integration of non-replicating vectors into the genome.
[41] Multiple variants of loxP,[42] in particular lox2272 and loxN, have been used by researchers with the combination of different Cre actions (transient or constitutive) to create a "Brainbow" system that allows multi-colouring of mice's brain with four fluorescent proteins.
