Recombinase-mediated cassette exchange

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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).

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 unstable 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:

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 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 either by a standard transfection approach or by RMCE, respectively. 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 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 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 either by a standard transfection approach or by RMCE, respectively. 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

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 of a sequence that is flanked by two equally-oriented identical FRTs
  • the inefficient addition of an extra piece of DNA carrying a single FRT site that is identical to the target site

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:

  • 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 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. Please note that RMCE introduces just one copy of the GOI at the pre-determined locus ant that it does not co-introduce prokaryotic vector sequences (dotted lines) that would otherwise trigger immunologic or epigenetic defense mechanisms.

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.

[edit] RMCE-Multiplexing

Figure 2: RMCE multiplexing. 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 use 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
Figure 2: RMCE multiplexing. 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 use 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

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.


[edit] References

  • T. Schlake & J. Bode (1994), Biochemistry 33, 12746-12751. Use of mutated FLP-recognition-target-(FRT-)sites for the exchange of expression cassettes at defined chromosomal loci.
  • J. Bode, S. Götze, M. Klar, K. Maaß, K. Nehlsen, A. Oumard & S. Winkelmann (2004) BIOForum 34-36 Den Viren nachempfunden: Effiziente Modifikation von Säugerzellen.
  • F. Cesari, V. Rennekampff, K. Vintersten, L. G. Vuong, J. Seibler, J. Bode, F. F. Wiebel & A. Nordheim, A. (2004) Elk-1 knock-out mice engineered by Flp recombinase-mediated cassette exchange. Genesis 38, 87-92.
  • A. J. M. Roebroek, S. Reekmans, A. Lauwers, N. Feyaerts, L. Smeijers & D. Hartmann (2006). Mutant Lrp1 knock-in mice generated by RMCE reveal differential importance of the NPXY motifs in the intracellular domain of LRP1 for normal fetal development. Mol. Cell Biol. 26: 605–616

[edit] Review Articles

  • J. Bode, T. Schlake, M. Iber, D. Schübeler, J. Seibler, E. Snezhkov & L. Nikolaev (2000) The transgeneticist's toolbox - Novel methods for the targeted modification of eukaryotic genomes. Biol. Chem. 381, 801-813.
  • C.S. Branda & S.M. Dymecki (2004) Talking about a revolution: the impact of site-specific recombinases on genetic analyses in mice. Develop. Cell 6, 7-28.
  • A. Oumard, J. Qiao, T. Jostock, J. Li & J. Bode (2006): Recommended Method for Chromosome Exploitation: RMCE-based Cassette-Exchange Systems in Animal Cell Biotechnology. In: Cytotechnology. Bd. 50, Nr. 1-3, S. 93-108. doi:10.1007/s10616-006-6550-0
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