Recombinase-Mediated Cassette Exchange (RMCE): Traditional Concepts and Current Challenges

• Published in: Journal of molecular biology Vol. 407; no. 2; pp. 193 - 221
• Main Authors: Turan, Soeren, Galla, Melanie, Ernst, Ellen, Qiao, Junhua, Voelkel, Christine, Schiedlmeier, Bernhard, Zehe, Christoph, Bode, Juergen
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 25.03.2011
• Subjects: Animals; DNA Nucleotidyltransferases - genetics; Flp-RMCE; Gene Targeting; Gene Transfer Techniques; Genetic Engineering - methods; integrase mechanism; Mice; recombinase-mediated cassette exchange; tag and exchange; targeted integration; Transgenes; recombinase-mediated cassette exchange; CPP; heteromeric RTs; RT; RV; LE/RE; pFARs; FBE; FRT; integrase mechanism; HR; ES cells; FACS; DSB; NLS; tag and exchange; targeted integration; hygtk; GOI; dRMCE; RMDI; loxP; iPS; Flp-RMCE; SSR; heterospecific RTs; ZFNs; RMCE
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2011.01.004

Traditional DNA transduction routes used for the modification of cellular genomes are subject to unpredictable alterations, as the cell-intrinsic repair machinery may affect both the integrity of the transgene and the recipient locus. These problems are overcome by recombinase-mediated cassette exchange (RMCE) approaches enabling predictable expression patterns by the nondisruptive insertion of a gene cassette at a pre-characterized genomic locus. The destination is marked by a “tag” consisting of two heterospecific recombination target sites (RTs) at the flanks of a selection marker. Provided on a circular donor vector, an analogous cassette encoding the gene of interest can cleanly replace the resident cassette under the influence of a site-specific recombinase. RMCE was first based on the yeast integrase Flp but had to give way to the originally more active phage-derived Cre enzyme. To be effective, both Tyr-recombinases have to be applied at a considerable concentration, which, in the case of Cre, triggers endonucleolytic activities and therefore cellular toxicity. This review addresses the particularities of both recombination routes depending on the structure of the synaptic complex and on improved integrase and RT variants. While the performance of Flp-RMCE can now firmly rely on optimized Flp variants and multiple sets of functional target sites (FRTs), the Cre system suffers from the promiscuity of its RT mutants, which is explained in molecular terms. At present, RMCE enters applications in the stem cell field. Remarkable efforts are noted in the framework of various mouse mutagenesis programs, which, in their first phase, have targeted virtually all genes and now start to shift their emphasis from gene trapping to gene modification. [Display omitted]