Recombinase-mediated cassette exchange

RMCE (recombinase-mediated cassette exchange) is a procedure in reverse genetics allowing the systematic, repeated modification of higher eukaryotic genomes by targeted integration, based on the features of site-specific recombination processes (SSRs). For RMCE, this is achieved by the clean exchange of a preexisting gene cassette for an analogous cassette carrying the 'gene of interest' (GOI). RMCE (recombinase-mediated cassette exchange) is a procedure in reverse genetics allowing the systematic, repeated modification of higher eukaryotic genomes by targeted integration, based on the features of site-specific recombination processes (SSRs). 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 This spectrum of options could be extended significantly by the generation of spacer mutants for extended 48 bp FRT sites (cross-hatched half-arrows in Figure 1). Each mutant Fn recombines with an identical mutant Fn with an efficiency equal to the wildtype sites (F x F). A cross-interaction (F x Fn) is strictly prevented by the particular design of these components. This sets the stage for the situation depicted in Figure 1A: First applied for the Tyr-recombinase Flp, this novel procedure is not only relevant to the rational construction of biotechnologically significant cell lines, but it also finds increasing use for the systematic generation of stem cells. Stem cells can be used to replace damaged tissue or to generate transgenic animals with largely pre-determined properties.