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).
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 has been previously established that coexpression of both Cre and Flp recombinases catalyzes the exchange of sequences flanked by single loxP and FRT sites integrated into the genome at a random location.
However, these studies did not explore whether such an approach could be used to modify conditional mouse alleles carrying single or multiple loxP and FRT sites.
The correctly replaced locus would encode the custom modification and a different drug-selection cassette flanked by single loxP and FRT sites.
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.
Figure 1: Principle of RMCE:
exchange of genetic cassettes (´flip´ step) is enabled by a
recombinase
(´Flp´) from yeast. Part B shows mutants (Fn) of the naturally occurring 48 bp
FRT
-site (F). If a gene cassette is flanked by a set of these sites (F and Fn, for example) it can change places, by double-reciprocal recombination, with a second cassette that is part of an exchange plasmid (Figure 1, part A). A model experiment is shown in part C, in which an ´empty´ cell is modified by either a standard
transfection
approach or by RMCE. Please note that in the first case multiple genomic sites are hit, each giving raise to a different expression level (cf. the broad distribution of green dots). If a pre-defined genomic address is used to introduce the same gene reporter, each
clone
derived from such an event shows comparable expression characteristics
Figure 2: Multiplexing RMCE
. In the given example a reporter gene cassette (gfp/tk/neo), flanked by four heterospecific
FRT-
sites (F5/F3-F/Fn) is introduced into the genome. The unique F5/F3 address can then be used to introduce an upstream-regulatory element and the F/Fn address to apply a similar modification at the downstream end. after the expression of the gfp-reporter has been optimized by systematic changes of this type, the central reporter cassette can be exchanged for any ´gene-of-interest´(GOI): the GOI will be flanked by the F3 and F-sites, respectively and introduced accordingly while the flanking elements will remain in place
