Multiplexing RMCE
Multiplexing setups rely on the fact that each F-Fn pair (consisting of a wildtype FRT site and a mutant called "n") or each Fn-Fm pair (consisting of two mutants, "m" and "n") constitutes a unique "address" in the genome. A prerequisite are differences in four out of the eight spacer positions (see Figure 1B). If the difference is below this threshold, some cross-interaction between the mutants may occur leading to a faulty deletion of the sequence between the heterospecific (Fm/Fn or F/Fn) sites.
13 FRT-mutants have meanwhile become available, which permit the establishment of several unique genomic addresses side-by side (for instance F-Fn and Fm-Fo). These addresses will be recognized by donor plasmids that have been designed according to the same principles, permitting successive (but also synchonous) modifications at the predetermined loci. These modifications can be driven to completion in case the compatible donor plasmid(s) are provided at an excess (mass-action principles). Figure 2 illustrates one use of the multiplexing principle: the stepwise extension of a coding region in which a basic expression unit is provided with genomic insulators, enhancers, or other cis-acting elements.
A recent variation of the general concept is based on PhiC31 (an integrase of the Ser-class), which permits introduction of another RMCE target at a secondary site after the first RMCE-based modification has occurred. This is due to the fact that each phiC31-catalyzed exchange destroys the attP and attB sites it has addressed converting them to attR and attL product sites, respectively. While these changes permit the subsequent mounting of new (and most likely remote) targets, they do not enable addressing several RMCE targets in parallel, nor do they permit "serial RMCE", i.e. successive, stepwise modifications at a given genomic locus.
It should be noted that this is different for Flp-RMCE, for which the post-RMCE status of FRTs corresponds to their initial state. This property enables the intentional, repeated mobilization of a target cassette by the addition of a new donor plasmid with compatible architecture. These "multiplexing-RMCE" options open unlimited possibilities for serial- and parallel specific modifications of pre-determined RMCE-targets
Read more about this topic: Recombinase-mediated Cassette Exchange