Methods
Gene targeting methods are established for several model organisms and may vary depending on the species used. In general, a targeting construct made out of DNA is generated in bacteria. It typically contains part of the gene to be targeted, a reporter gene, and a (dominant) selectable marker.
To target genes in mice, this construct is then inserted into mouse embryonic stem cells in culture. After cells with the correct insertion have been selected, they can be used to contribute to a mouse's tissue via embryo injection. Finally, chimeric mice where the modified cells made up the reproductive organs are selected for via breeding. After this step the entire body of the mouse is based on the previously selected embryonic stem cell.
To target genes in moss, this construct is incubated together with freshly isolated protoplasts and with Polyethylene glycol. As mosses are haploid organisms, regenerating moss filaments (protonema) can directly be screened for gene targeting, either by treatment with antibiotics or with PCR. Unique among plants, this procedure for reverse genetics is as efficient as in yeast. Using modified procedures, gene targeting has also been successfully applied to cattle, sheep, swine, and many fungi.
The frequency of gene targeting can be significantly enhanced through the use of engineered endonucleases such as zinc finger nucleases, engineered homing endonucleases, and nucleases based on engineered TAL effectors. To date, this method has been applied to a number of species including Drosophila melanogaster, tobacco, corn, human cells, mice, and rats.
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