Int J Mol Sci
The site-specific recombination technologies is a gene engineering tool that relies on recombinases (Cre, FLP) to replace specific DNA sequences.
They precisely recognize DNA sites of 30–50 bp (lox, FRT), thereby bringing them together, cutting, swapping and recombining the DNA in a new configuration, which typically results in gene insertions, deletions or inversions.
This technology is particularly powerful when a mutation needs to be restricted to certain time-points in development and/or specific cell types (conditional models).
Nowadays, the most commonly used systems for cell line and rodent gene engineering are the Cre-lox and FLP-FRTsystems.
The recombinase protein (Cre, FLP) recognizes and binds two DNA sites (lox, FRT), bringing them together to form a complex. It is within this complex where the DNA gets cut and recombined.
When the two sites are on the same molecule, the outcome will be either an excision or inversion, depending on whether the two sites are in the same or opposite orientation, respectively.
An insertion occurs when the recognition sites are located on two different DNA molecules, with one being circular.
SSR-based genome modification strategies
Besides the systematic use of Cre-loxP and FLP-FRT systems for conditional modifications (e.g., tissue specificity) or the excision of selection makers, three successful genetic strategies are worth mentioning in greater detail:
The temporal control of Cre activity allows the induction of genetic modifications later in embryogenesis or adult animals. This bypasses potential drawbacks such as embryonic lethality.
To specifically induce temporal Cre activity, the recombinase is fused with a mutated form of the ligand-binding domain of the human estrogen receptor (ERt2). The estrogen receptor antagonist tamoxifen binds Cre-ERt2 and allows the penetration of the complex into the nucleus where Cre induces site-specific gene modification.
In absence of tamoxifen, the Cre-ERt2 fusion protein remains strictly cytoplasmic.
This strategy is an elegant way to spatially induce a mutation and the reference technology to activate point-mutations under control of the endogenous promoter of the wildtype gene.
The site-specific recombinase (Cre or FLP) switches on the genetic modification by inverting, and thereby activating, the DNA sequence of interest.
Recombinase-mediated cassette exchange (RMCE) is the preferred strategy for inserting large genomic sequences. It also allows the generation of several mutants from the same parental embryonic stem cell clone for comparative studies.
The wildtype gene is flanked by two different specific sites. In the presence of a DNA plasmid holding the mutated form of the gene of interest flanked by the same two different specific recognition sites, a recombinase like Cre can exchange/swap the wildtype gene with the mutated variant.
Int J Mol Sci
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