Recombinase-mediated cassette exchange

RMCE (English recombinase -mediated cassette exchange) is a method of reverse genetics and is used for systematic modification in eukaryotic cells through targeted exchange of gene cassettes.

Motivation

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

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

Principle

This is where the RMCE cassette exchange method, which provides vectors with tools of yeast:

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

• An inversion ( inversion of the two oppositely oriented FRT sites boxed sequence)
• A deletion (also called " excision " or called "Resolution", loss of one of two rectified FRTS boxed sequence) or, in the reverse of this process,
• An inefficient addition (also called " integration "; union of two DNA segments carrying the same type FRT sites ).

1994 was the spectrum of possibilities to be expanded. ( Hatched half- arrows in the figure Fn ) of these FRT sites (F) prepared with one another as efficiently recombine like two wild-type sites for this purpose mutants. One thing they do not show: a Kreuzrekombination the type F x Fn, which would lead to deletion. Thus we find ourselves in the situation of Figure:

• Any gene (in this case a composite selection gene ) is enclosed by a set of FRT sites, Fn and F; after its introduction into the genome of the host cell, the characteristics of different integration sites and are characterized particularly suitable clones are isolated;

• The gene is actually of interest is cloned (one red and one half- hatched arrow) between a corresponding set of Fn and F bodies and transferred as part of a circular vector into the host cell. Flp recombinase is now being used to a double -reciprocal ' crossover ' to induce. This procedure conducted according to the " RMCE cassette exchange principle" the gene of interest exactly at the predetermined location - a process that can be repeated any number of times with different constructs.

This new method not only arrives for the rational design of biotechnologically important cell lines meaning, but is also increasingly being used for the systematic modification of stem cells, such as with the intention of deliberately creating transgenic animals.