Gene targeting
As gene targeting ( English gene targeting , German and gene targeting , targeted gene modification ) is in the genetics refers to a technique that the homologous recombination exploit to an endogenous gene to be changed. The method can be used to delete genes , remove exons or introduce other gene mutations . Gene targeting can be permanent, but it can also be conditionally regulated, depending on certain conditions, induced. The conditions can be time-dependent on the stage of development , or also depending on the tissue . It is necessary to create a specific vector for each gene that comes into question (targeting vector). Gene targeting can be applied to any gene, regardless of transcriptional activity or gene size. Mario R. Capecchi , Martin J. Evans and Oliver Smithies received the 2007 Nobel Prize in Physiology and Medicine for their “discoveries on the basis for inducing specific gene modifications in mice with the help of embryonic stem cells”.
method
Gene targeting methods depend on the model organism . With genes in mice - roughly speaking - the following steps are necessary (see also under knockout mouse ): The creation of a target construct from DNA that is amplified in bacteria - this typically contains a part of the gene that is to be hit, a selectable marker and often a reporter gene . This construct is then introduced into embryonic stem cells that are used in a cell culture . After the cells with the correct insertion have been selected by selection and analysis using Southern blot analysis or PCR , they can be used to contribute to the tissue of a mouse by injecting them into an embryo . Chimeric mice are selected for breeding based on their coat color . If the modified cells participate in the germ line, mating with wild-type animals results in heterozygous animals in which one allele carries the gene change. After several backcrosses to a suitable strain, heterozygous animals are crossed with one another. In accordance with Mendel's rules , offspring are expected who have the wild-type genome, or are heterozygous or homozygous for the changed allele .
In order to specifically change genes in mosses (see also knockout moss ), this DNA construct is incubated with protoplasts and with polyethylene glycol. Since mosses are haploid organisms , regenerating moss filaments ( protonemes ) can be checked directly for gene targeting, for example within just 6 weeks using PCR methods. In contrast to vascular plants , this reverse genetics process is as efficient in the deciduous moss Physcomitrella patens as it is in yeast .
The change in DNA takes place e.g. B. by the Cre / loxP system or by the Flp / FRT system ( RMCE cartridge exchange method ).
Comparison with gene trapping
Gene trapping is based on the random integration of an altered nucleotide sequence into the genome , while gene targeting integrates the altered nucleotide sequence into a specific region in the genome.
literature
- Jochen Graw (2007): Nobel Prize 2007 in Medicine: Making knockout mice. In: Biology in Our Time. 37 (6): 352-354. PDF
Web links
- Outline of gene targeting (University of Michigan)
- Targeted gene modification in barley
Individual evidence
- ↑ Information from the Nobel Foundation on the 2007 award ceremony for Mario Capecchi, Martin Evans and Oliver Smithies (English)
- ^ Ralf Reski (1998): Physcomitrella and Arabidopsis : the David and Goliath of reverse genetics . In: Trends in Plant Science. 3: 209-210. doi: 10.1016 / S1360-1385 (98) 01257-6