Recombinase-mediated cassette exchange

In the field of reverse genetics RMCE (recombinase-mediated cassette exchange) is of increasing relevance. Based on the features of site-specific recombination processes (SSRs), the procedure permits the systematic, repeated modification of higher eukaryotic genomes by targeted integration. For RMCE, this is achieved by the clean exchange of a preexisting gene cassette for an analogous cassette carrying the "gene of interest" (GOI).

The genetic modification of mammalian cells is a standard procedure for the production of correctly modified proteins with pharmaceutical relevance. To be successful, the transfer and expression of the transgene has to be highly efficient and should have a largely predictable outcome. Current developments in the field of gene therapy are based on the same principles. Traditional procedures used for transfer of GOIs are not sufficiently reliable, mostly because the relevant epigenetic influences have not been sufficiently explored: transgenes integrate into chromosomes with low efficiency and at loci that provide only sub-optimal conditions for their expression. As a consequence the newly introduced information may not be realized (expressed), the gene(s) may be lost and/or re-insert and they may render the target cells in unstable state. It is exactly this point where RMCE enters the field. The procedure was introduced in 1994 and it uses the tools yeasts and bacteriophages have evolved for the efficient replication of important genetic information:

Most yeast strains contain circular, plasmid-like DNAs called "two-micron circles". The persistence of these entities is granted by a recombinase called "flippase" or "Flp". Four monomers of this enzyme associate with two identical short (48 bp) target sites, called FRT ("flip-recombinase targets"), resulting in their crossover. The outcome of such a process depends on the relative orientation of the participating FRTs leading to

...
Wikipedia