Recombinase-mediated Cassette Exchange - General Principles

General Principles

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

• the inversion of a sequence that is flanked by two identical but inversely-oriented FRT sites
• the deletion/resolution of a sequence that is flanked by two equally-oriented identical FRTs
• the inefficient reversion of the letter process, commonly called integration or "addition" of an extra piece of DNA carrying a single FRT site identical to the target site

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:

• a target cassette (here a composite +/- selection marker) is flanked by an F- and an Fn site. After its introduction into the genome of a host cell the properties of many integration sites (genomic ´addresses´) are characterized and appropriate clones are isolated
• the GOI (gene-of-interest) is part of a circular ´exchange plasmid´ and is flanked by a set of matching sites. This exchange plasmid can be introduced into the cell at large molecular excess and will thereby undergo the depicted exchange (RMCE-) reaction with the pre-selected genomic address (i.e. the F <+/-> Fn target)
• this RMCE-principle is a process that can be repeated with the same or a different exchange plasmid ("serial RMCE"). Please note that RMCE introduces just one copy of the GOI at the pre-determined locus and that it does not co-introduce prokaryotic vector sequences (dotted lines) that would otherwise trigger immunologic or epigenetic defense mechanisms.

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.