Search tips
Search criteria 


Logo of cytotechspringer.comThis journalToc AlertsSubmit OnlineOpen Choice
Cytotechnology. 2006 March; 50(1-3): 93–108.
Published online 2006 June 14. doi:  10.1007/s10616-006-6550-0
PMCID: PMC3476001

Recommended Method for Chromosome Exploitation: RMCE-based Cassette-exchange Systems in Animal Cell Biotechnology


The availability of site-specific recombinases has revolutionized the rational construction of cell lines with predictable properties. Early efforts were directed to providing pre-characterized genomic loci with a single recombinase target site that served as an address for the integration of vectors carrying a compatible tag. Efficient procedures of this type had to await recombinases like ΦC31, which recombine attP and attB target sites in a one-way reaction — at least in the cellular environment of the higher eukaryotic cell. Still these procedures lead to the co-introduction of prokaryotic vector sequences that are known to cause epigenetic silencing. This review illuminates the actual status of the more advanced recombinase-mediated cassette exchange (RMCE) techniques that have been developed for the major members of site-specific recombinases (SR), Flp, Cre and ΦC31. In RMCE the genomic address consists of a set of heterospecific recombinase target (RT-) sites permitting the exchange of the intervening sequence for the gene of interest (GOI), as part of a similar cassette. This process locks the GOI in place and it is ‘clean’ in the sense that it does not co-introduce prokaryotic vector parts nor does it leave behind a selection marker.

Key words: Cassette exchange, Flexing, Floxing, Flrting, Froxing, RMCE, Site-specific recombinase, Tissue engineering


Flp-recombinase target site
homologous recombination
recombinase-mediated cassette exchange
recombinase target
site-specific recombination


  • Andreas S., Schwenk F., Küter-Luks B., Faust N., Kühn R. Enhanced efficiency through nuclear localization signal fusion on phage C31-integrase: activity comparison with Cre and FLPe recombinase in mammalian cells. Nucleic Acids Res. 2002;30:2299–2306. doi: 10.1093/nar/30.11.2299. [PMC free article] [PubMed] [Cross Ref]
  • Baer A., Schübeler D., Bode J. Transcriptional properties of genomic transgene integration sites marked by electroporation or retroviral infection. Biochemistry. 2000;39:7041–7049. doi: 10.1021/bi992957o. [PubMed] [Cross Ref]
  • Baer A., Bode J. Coping with kinetic and thermodynamic barriers: RMCEan efficient strategy for the targeted integration of transgenes. Curr. Opin. Biotech. 2001;12:473–480. doi: 10.1016/S0958-1669(00)00248-2. [PubMed] [Cross Ref]
  • Baer A. 2002. Funktioneller Vergleich von S/MARs (‘scaffold/matrix attachment regions’ und Insulatoren im chromosomalen Kontext. Dissertation, University of Braunschweig;
  • Bautista D., Shulman M.J. A hit-and-run system for introducing mutations into the Ig H chain locus of hybridoma cells by homologous recombination. J. Immunol. 1993;151:1950–1958. [PubMed]
  • Belteki G., Gertsenstein M., Ow D.W., Nagy A. Site-specific cassette exchange and germline transmission with mouse ES cells expressing phiC31 integrase. Nat. Biotechnol. 2003;21:321–324. doi: 10.1038/nbt787. [PubMed] [Cross Ref]
  • Bode J., Bartsch J., Boulikas T., Iber M., Mielke C., Schübeler D., Seibler J. and Benham C. 1998. Transcription-promoting genomic sites in mammalia: their elucidation and architectural principles. Gene Ther. Mol. Biol. 1: 551–880.
  • Bode J., Schlake T., Iber M., Schübeler D., Seibler J., Snezhkov E., Nikolaev L. The transgeneticist’s toolbox — novel methods for the targeted modification of eukaryotic genomes. Biol. Chem. 2000;381:801–813. doi: 10.1515/BC.2000.103. [PubMed] [Cross Ref]
  • Bode J., Goetze S., Ernst E., Huesemann Y., Baer A., Seibler J., Mielke C. Architecture and utilization of highly-expressed genomic sites in New Comprehensive Biochemistry Vol 38. In: Makrides S., Bernardi G., editors. Gene Transfer and Expression in Mammalian Cells. Amsterdam: Elsevier; 2003.
  • Bode J., Winkelmann S., Götze S., Spiker S., Tsutsui K., Bi C. and Benham C. 2005. Correlations between scaffold/matrix attachment region (S/MAR) binding activity and DNA duplex destabilization energy. J. Mol. Biol. in press. [PubMed]
  • Bouhassira E., Westerman K., Leboulch P. Transcriptional behavior of LCR enhancer elements integrated at the same chromosomal locus by recombinase-mediated cassette exchange. Blood. 1997;90:3332–3344. [PubMed]
  • Branda C.S., Dymecki S.M. Talking about a revolution: the impact of site-specific recombinases on genetic analyses in mice. Develop. Cell. 2004;6:7–28. doi: 10.1016/S1534-5807(03)00399-X. [PubMed] [Cross Ref]
  • Buchholz F., Ringrose L., Angrand P., Rossi F., Stewart A. Different thermostabilities of FLP and Cre recombinases: implications for applied site specific recombination. Nucl. Acids Res. 1996;24:4256–4262. doi: 10.1093/nar/24.21.4256. [PMC free article] [PubMed] [Cross Ref]
  • Buchholz F., Angrand P.-O., Stewart A.F. Improved properties of Flp recombinase evolved by cycling mutagenesis. Nat. Biotechnol. 1998;16:657–662. doi: 10.1038/nbt0798-657. [PubMed] [Cross Ref]
  • Cesari F., Rennekampff V., Vintersten K., Vuong L.G., Seibler J., Bode J., Wiebel F.F., Nordheim A. Elk-1 knock-out mice engineered by Flp recombinase-mediated cassette exchange. Genesis. 2004;38:87–92. doi: 10.1002/gene.20003. [PubMed] [Cross Ref]
  • Cobellis G., Nicolaus G., Iovino M., Romito A., Marra E., Barbarisi M., Sardiello M., Di Giorgio F.P., Iovino N., Zollo M., Ballabio A., Cortese R. Tagging genes with cassette-exchange sites. Nucl. Acids Res. 2005;33(4):e44. doi: 10.1093/nar/gni045. [PMC free article] [PubMed] [Cross Ref]
  • Coroadinha A.S., Schucht R., Gama-Norton L., Wirth D., Hauser H. and Carrondo M.J.T. 2005. The use of recombinase cassette exchange in retroviral vector producer cell lines: predictability and efficiency in transgene replacement. J. Biotechnol., Submitted. [PubMed]
  • Fukushige S., Sauer B. Genomic targeting with a positive-selection lox integration vector allows highly reproducible gene expression in mammalian cells. Proc. Natl. Acad. Sci. 1992;89:7905–7909. doi: 10.1073/pnas.89.17.7905. [PubMed] [Cross Ref]
  • Garrick D., Fiering S., Martin D.I.K., Whitelaw E. Repeat-induced gene silencing in mammals. Nat. Genet. 1998;18:56–59. doi: 10.1038/ng0198-56. [PubMed] [Cross Ref]
  • Goetze S., Huesemann Y., Baer A., Bode J. Functional characterization of transgene integration patterns by Halo-FISH: electroporation versus retroviral infection. Biochemistry. 2003;42:7035–7043. doi: 10.1021/bi0340907. [PubMed] [Cross Ref]
  • Goetze S., Baer A., Winkelmann S., Nehlsen K., Seibler J., Maass K., Bode J. Genomic bordering elements: their performance at pre defined genomic loci. Mol. Cell. Biol. 2005;25:2260–2272. doi: 10.1128/MCB.25.6.2260-2272.2005. [PMC free article] [PubMed] [Cross Ref]
  • Grabenhorst E., Schlenke P., Pohl S., Nimtz M., Conradt H.S. Genetic engineering of recombinant glycoproteins and the glycosylation pathway in mammalian host cells. Glycoconjugate J. 1999;16:81–97. doi: 10.1023/A:1026466408042. [PubMed] [Cross Ref]
  • Groth A.C., Olivares E.C., Thyagarajan B., Calos M.P. A phage integrase directs efficient site-specific integration in human cells. Proc. Natl. Acad. Sci. USA. 2000;97:5995–6000. doi: 10.1073/pnas.090527097. [PubMed] [Cross Ref]
  • Kolot M., Silberstein N., Yagil E. Site specific recombination in mammalian cells expressing the Int recombinase of bacteriophage HK022. Mol. Biol. Rep. 1999;26:207–213. doi: 10.1023/A:1007096701720. [PubMed] [Cross Ref]
  • Kouzine K., Liu L., Sanford S., Chung H.J., Levens D. The dynamic response of upstream DNA to transcription-generated torsional stress. Nature Struct. Mol. Biol. 2004;11:1092–1100. doi: 10.1038/nsmb848. [PubMed] [Cross Ref]
  • Lauth M., Spreafico F., Dethleffsen K., Meyer M. Stable and efficient cassette exchange under non-selectable conditions by combined use of two site-specific recombinases. Nucl. Acids Res. 2002;30:e115. doi: 10.1093/nar/gnf114. [PMC free article] [PubMed] [Cross Ref]
  • Mlynarova L., Libantova J., Vrba L., Nap J.P. The promiscuity of heterospecific lox sites increases dramatically in the presence of palindromic DNA. Gene. 2002;296:129–137. doi: 10.1016/S0378-1119(02)00841-7. [PubMed] [Cross Ref]
  • Nakano M., Odaka K., Takahashi Y., Ishimura M., Saito I., Kanegae Y. Production of viral vectors using recombinase-mediated cassette exchange. Nucl. Acids Res. 2005;33:e76. doi: 10.1093/nar/gni074. [PMC free article] [PubMed] [Cross Ref]
  • Ng P., Baker M.D. High efficiency site-specific modification of the chromosomal immunoglobulin locus by gene targeting. J. Immunol. Meth. 1998;214:81–96. doi: 10.1016/S0022-1759(98)00033-7. [PubMed] [Cross Ref]
  • O’Gorman S., Fox D.T., Wahl G.M. Recombinase-mediated gene activation and site-specific integration in mammalian cells. Science. 1991;251:1351–1355. doi: 10.1126/science.1900642. [PubMed] [Cross Ref]
  • Peitz M., Pfannkuche K., Rajewsky K., Edenhofer F. Ability of the hydrophobic FGF and basic TAT peptides to promote cellular uptake of recombinant Cre recombinase: a tool for efficient genetic engineering of mammalian genomes. Proc. Natl. Acad. Sci. USA. 2002;99:4489–4494. doi: 10.1073/pnas.032068699. [PubMed] [Cross Ref]
  • Roebroek A.J.M., Reekmans S., Lauwers A., Feyaerts N., Smeijers L. and Hartmann D. 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 [PMC free article] [PubMed]
  • Riu E., Grimm D., Huang Z., Mark A., Kay M.A. Increased maintenance and persistence of transgenes by excision of expression cassettes from plasmid sequences in vivo. Hum. Gene. Ther. 2005;16:558–570. doi: 10.1089/hum.2005.16.558. [PubMed] [Cross Ref]
  • Schlake T., Bode J. Use of mutated FLP recognition target (FRT) sites for the exchange of expression cassettes at defined chromosomal loci. Biochemistry. 1994;33:12746–12751. doi: 10.1021/bi00209a003. [PubMed] [Cross Ref]
  • Schmidt E.E., Taylor D.S., Prigge J.R., Arnett S., Capecchi M.R. Illegitimate Cre-dependent chromosome rearrangements in transgenic mouse spermatids. Proc. Natl. Acad. Sci. USA. 2000;97:13702–13707. doi: 10.1073/pnas.240471297. [PubMed] [Cross Ref]
  • Schnütgen F., Doerflinger N., Calléja C., Wendling O., Chambon P., Ghyselinck N.B. A directional strategy for monitoring Cre-mediated recombination at the cellular level in the mouse. Nat. Biotechnol. 2003;21:562–565. doi: 10.1038/nbt811. [PubMed] [Cross Ref]
  • Schucht R., Coroadinha A.S., Zanta-Boussif M.A., Carrondo M., Hauser H. and Wirth D. 2005. A new generation of retroviral producer cells: predictable and stable virus production by Flp mediated site-specific integration of retroviral vectors. Mol. Thera., Submitted. [PubMed]
  • Schübeler D., Bode J. Retargeting of retroviral integration sites for the predictable expression of transgenes and the analysis of cis-acting sequences. Biochemistry. 1998;37:11907–11914. doi: 10.1021/bi9807052. [PubMed] [Cross Ref]
  • Seibler J., Schübeler D., Fiering S., Groudine M., Bode J. DNA cassette exchange mediated by FLP recombinase: an efficient strategy for the repeated modification of tagged loci by marker-free constructs. Biochemistry. 1998;37:6229–6234. doi: 10.1021/bi980288t. [PubMed] [Cross Ref]
  • Seibler J., Küter-Luks B., Kern H., Streu S., Plum L., Mauer J., Kühn R., Brüning J.C., Schwenk F. Single copy shRNA configuration for ubiquitous gene knockdown in mice. Nucl. Acids Res. 2005;33:e67. doi: 10.1093/nar/gni065. [PMC free article] [PubMed] [Cross Ref]
  • Stark W.M., Boocock M.R., Sherratt D.J. Catalysis by site-specific recombinases. Trends Genet. 1992;8:432–439. doi: 10.1016/0168-9525(92)90176-5. [PubMed] [Cross Ref]
  • Taniguchi M., Sanbo M., Watanabe S., Naruse I., Mishina M., Yagi T. Efficient production of Cre-mediated site-directed recombinants through the utilization of the puromycin resistance genepac: a transient gene-integration marker for ES cells. Nucl. Acids Res. 1998;26:679–680. doi: 10.1093/nar/26.2.679. [PMC free article] [PubMed] [Cross Ref]
  • Thorpe H.M., Smith M.C.M. In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family. Proc. Natl. Acad. Sci. USA. 1998;95:5505–5510. doi: 10.1073/pnas.95.10.5505. [PubMed] [Cross Ref]
  • Thyagarajan B., Olivares E.C., Hollis R.P., Ginsburg D.S., Calos M.P. Site-specific genomic integration in mammalian cells mediated by phage phiC31 integrase. Mol. Cell Biol. 2001;21:3926–3934. doi: 10.1128/MCB.21.12.3926-3934.2001. [PMC free article] [PubMed] [Cross Ref]
  • Umlauf S.W., Cox M.M. The functional significance of DNA sequence structure in a site-specific genetic recombination reaction. EMBO J. 1988;7:1845–1852. [PubMed]
  • Unsinger J., Lindenmaier W., May T., Hauser H., Wirth D. Stable and strictly controlled expression of LTR-flanked autoregulated expression cassettes upon adenoviral transfer. Biochem. Biophys. Res. Commun. 2004;319:879–887. doi: 10.1016/j.bbrc.2004.05.067. [PubMed] [Cross Ref]
  • Weidle U.H., Buckel P., Wienberg J. Amplified expression constructs for human tissue-type plasminogen activator in Chinese hamster ovary cells: instability in the absence of selective pressure. Gene. 1988;66:193–203. doi: 10.1016/0378-1119(88)90356-3. [PubMed] [Cross Ref]
  • Winkler K., Wermelinger T., Paul C., Koch S., Brecht S., Zietze S., Nuck R., Thiel G., Marx U. and Sandig V. 2005. Targeting the human IgH loci for high level heterologous gene expression. In: Gòdia F. and Fussenegger M.(eds) ESACT Proceedings, Vol. 2. Animal Cell Technology Meets Genomics. SpringerNetherlands, pp.403–409.ISBN: 1-4020-2791-5
  • Wirth M., Bode J., Zettelmeissl G., Hauser H. Isolation of overproducing recombinant mammalian cell lines by a fast and simple selection procedure. Gene. 1988;73:419–426. doi: 10.1016/0378-1119(88)90506-9. [PubMed] [Cross Ref]
  • Wirth D., Hauser H. Flp-mediated integration of expression cassettes into FRT-tagged chromosomal loci in mammalian cells. Methods Mol. Biol. 2004;267:467–476. [PubMed]
  • Wong E.T., Kolman J.L., Li Y.C., Mesner L.D., Hillen W., Berens C., Wahl G.M. Reproducible doxycycline-inducible transgene expression at specific loci generated by Cre-recombinase mediated cassette exchange. Nucl. Acids Res. 2005;33(17):e147. doi: 10.1093/nar/gni145. [PMC free article] [PubMed] [Cross Ref]
  • Wurm F.M., Jordan M. Gene transfer and gene amplification in mammalian cells in “New Comprehensive Biochemistry 38” In: Bernardi G., editor. A Gene Transfer and Expression in Mammalian Cells, Chapter 7. S. Makrides, Volume Ed. Amsterdam: Elsevier; 2003. pp. 307–335.

Articles from Cytotechnology are provided here courtesy of Springer Science+Business Media B.V.