Gene-Targeting

As gene targeting ( gene targeting English, German and gene targeting, targeted gene modification ) is referred to in genetics, a technique that takes advantage of homologous recombination to change an endogenous gene. The method can be used to delete genes to remove exons or to introduce other mutations. Gene targeting can be permanent and also depend on certain conditions. The conditions may at times depending on the development stage, or be dependent on the tissue. It requires the creation of a specific vector for each gene, which comes into question ( targeting vector ). Gene targeting can be used for any gene, regardless of the transcription activity, or the gene size. Mario R. Capecchi, Martin J. Evans and Oliver Smithies received the 2007 Nobel Prize for Physiology and Medicine for their "discoveries to the basics to induce specific gene modifications in mice using embryonic stem cells".

Method

Gene - targeting methods depend on the model organism. Are in genes in mice - roughly speaking - the following steps are required (see also knockout mouse ): the creation of a target construct of DNA, which is produced within bacteria - this typically includes a portion of the gene that is to be taken, a reporter and a selectable marker. Subsequently, this construct is introduced into embryonic stem cells are used in cell culture. After the cells have been selected with the correct insertion, they may be used to contribute to the tissue of a mouse by injecting an embryo. Finally, chimeric mice in which the reproductive organs consist of the modified cells are selected for breeding. Thereafter, the cells in the body of the mice based entirely on embryonic stem cell previously selected.

To specifically modify genes in mosses (see also knockout moss ), this DNA construct is incubated with protoplasts and polyethylene glycol. Since mosses are haploid organisms, regenerating Moosfilamente ( protonemata ) can be checked directly on gene targeting, for example, within just 6 weeks using PCR methods. Unlike vascular plants, this method of reverse genetics in the moss Physcomitrella patens as efficiently as in yeast.

The alteration of the DNA, for example by the Cre / loxP system or the FLP / FRT system ( RMCE cassette exchange procedure).

Comparison with Gene trapping

Gene trapping is based on the random integration of a modified nucleotide in the genome, while the modified nucleotide is incorporated into a specific region of the genome during gene targeting.