RMCE cartridge exchange procedure

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RMCE ( recombinase-mediated cassette exchange ) is a reverse genetics process and is used for the systematic modification of higher cells through the targeted exchange of gene cassettes.

motivation

The production of pharmaceutically important proteins is often achieved through the genetic modification of mammalian cells. This requires techniques of gene transfer and the efficient translation of genetic information ( expression ) in the target cell. Modern approaches to gene therapy are based on analogous principles.

Conventional methods of modifying animal cells are not very reliable, not least because the basic epigenetic principles are still too little known: smuggled genes only integrate sporadically into the genetic material of the host cell, and when they do, often in inappropriate places. As a result, the foreign gene is not or only irregularly read or remains inactive. To make matters worse, the newly incorporated genes are often detached from the host genome, and the cells become unstable.

principle

This is where the RMCE cartridge exchange process comes in, providing vectors with tools from the yeast:

RMCE principle: exchange of genetic cassettes ( flip ), made possible by the yeast RMCE recombinase ( Flp ). The inset shows mutants (Fn) of the naturally occurring FRT site (F) which, combined into sets (Fn, F; half-arrows), make the method possible. In the lower part of the inset, expression characteristics after conventional transfer (left) and based on RMCE (right) are shown.

In most yeast strains there is a structure called 2-micron circle , the survival of which is guaranteed by the recombinase Flp with unusual properties. Four monomeric molecules of this enzyme bind to two identical, short recognition sites (Flp-recombinase targets, FRT; red half arrows in the figure) and lead to their recombination ( crossover ). The result of this process is (depending on the relative orientation of the FRT sites)

  • an inversion (reversing the sequence framed by two oppositely oriented FRT sites),
  • a deletion (also called excision or resolution ; loss of a sequence framed by two FRTs in the same direction) or, in reverse of this process,
  • an inefficient addition (also integration ; union of two DNA segments that carry similar FRT sites).

In 1994 the spectrum of these possibilities was expanded. For this purpose, mutants (Fn; hatched half arrows in the figure) of these FRT sites (F) were produced, which recombine with one another as efficiently as two wild-type sites. There is one thing they do not show: a cross recombination of the type F x Fn, which would lead to deletion. So we are in the situation of the figure:

  • any gene (here a composite selection gene ) is framed by a set of FRT sites, Fn and F; after its introduction into the genome of the host cell, the properties of various integration sites are characterized and particularly suitable clones are isolated;
  • the gene that is actually of interest is cloned between a corresponding set of Fn and F positions (one red and one hatched half-arrow) and transferred into the host cell as part of a circular vector. Flp recombinase is now used to induce a double-reciprocal crossover . According to the RMCE cassette exchange principle, this procedure directs the gene of interest exactly to the predetermined location, a process that can be repeated as often as desired with different constructs.

This new method is not only important for the rational construction of biotechnologically important cell lines, but is also increasingly used for the systematic modification of stem cells, e.g. B. with the intention of specifically creating transgenic animals.

See also

literature

  • J. Bode, S. Götze, M. Klar, K. Maass, K. Nehlsen, A. Oumard, S. Winkelmann: Modeled on viruses: efficient modification of mammalian cells. In: BIOForum. 2004, pp. 34-36.
  • F. Cesari, V. Rennekampff, K. Vintersten, LG Vuong, J. Seibler, J. Bode, FF Wiebel, A. Nordheim: Elk-1 knock-out mice engineered by Flp recombinase-mediated cassette exchange. In: Genesis. 38, 2004, pp. 87-92.
  • J. Bode, T. Schlake, M. Iber, D. Schübeler, J. Seibler, E. Snezhkov, L. Nikolaev: The transgeneticists toolbox - Novel methods for the targeted modification of eukaryotic genomes. In: Biol. Chem. 381, 2000, pp. 801-813.
  • T. Schlake, J. Bode: Use of mutated FLP recognition target (FRT) sites for the exchange of expression cassettes at defined chromosomal loci. In: Biochemistry. 33, 1994, pp. 12746-12751.
  • AJM Roebroek, S. Reekmans, A. Lauwers, N. Feyaerts, L. Smeijers, D. Hartmann: 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. In: Mol. Cell Biol. 26, 2006, pp. 605-616.

Review article

  • A. Oumard, J. Qiao, T. Jostock, J. Li, J. Bode: Recommended Method for Chromosome Exploitation: RMCE-based Cassette-Exchange Systems in Animal Cell Biotechnology. In: Cytotechnology. Volume 50, No. 1-3, 2006, pp. 93-108. doi: 10.1007 / s10616-006-6550-0
  • S. Turan, J. Kuehle, A. Schambach, C. Baum, J. Bode: Multiplexing RMCE: Versatile Extensions of the Flp-Recombinase-Mediated Cassette-Exchange Technology. In: J. Mol. Biol. 402 (1), 2010, pp. 52-69. doi: 10.1016 / j.jmb.2010.07.015 . PMID 20650281
  • S. Turan, M. Galla, E. Ernst, J. Qiao, C. Voelkel, B. Schiedlmeier, C. Zehe, J. Bode: Recombinase-Mediated Cassette Exchange (RMCE): Traditional Concepts and Current Challenges. In: Journal of Molecular Biology . 407 (2), 2011, pp. 193-221. doi: 10.1016 / j.jmb.2011.01.004 . PMID 21241707
  • S. Turan, J. Bode: Site-specific recombinases: from tag-and-target- to tag-and-exchange-based genomic modifications. In: FASEB J . 25, 2011, pp. 4088-4107. doi: 10.1096 / fj.11-186940 .
  • S. Turan, C. Zehe, J. Kuehle, J. Qiao, J. Bode: Recombinase-Mediated Cassette Exchange (RMCE) - a rapidly-expanding toolbox for targeted genomic modifications. In: Genes. 515, 2013, pp. 1–27. doi: 10.1016 / j.gene.2012.11.016

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