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Recombinase-mediated cassette exchange
In the field of reverse genetics RMCE (recombinase-mediated cassette exchange) is of increasing relevance. 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).
Additional recommended knowledge
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. Actual approaches 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 principles 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 instable state.
It is exactly this point where RMCE enters the field. The procedure was introduced in 1994 and it uses the tools yeasts 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 FRT mutants (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:
This rather novel procedure is not only of relevance for the rational construction of biotechnologically relevant 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.
As stated above, each F-Fn pair (consisting of a wildtype FRT site and a mutant calle "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 positions in the spacer sequence(see Figure 1B). If the difference of spacer-composition 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.
Meanwhile 13 FRT-mutants (anticipated in Bode et al., 2000) are 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 targeting plasmids that have been designed
according to the same principles. A use of multiplexing is delineated in Figure 2: the stepwise extension of a coding region in which a basic expression unit is provided with genomic insulators, enhancers, or other cis-acting elements.
|This article is licensed under the GNU Free Documentation License. It uses material from the Wikipedia article "Recombinase-mediated_cassette_exchange". A list of authors is available in Wikipedia.|