Gene targeting

Gene targeting (also, replacement strategy based on homologous recombination) is a genetic technique that uses homologous recombination to change an endogenous gene. The method can be used to delete a gene, remove exons, and introduce point mutations. Gene targeting can be permanent or conditional. Conditions can be a specific time during development / life of the organism or limitation to a specific tissue, for example. Gene targeting requires the creation of a specific vector for each gene of interest. However, it can be used for any gene, regardless of transcriptional activity or gene size.

Method

Gene targeting methods vary depending on organism. To target genes in mice a rough outline of the necessary steps is as follows: First, 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. 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. Using modified procedures, gene targeting has also been successfully applied to cattle, sheep, swine, many fungi and mosses.

Comparison with gene trapping

Gene trapping is based on random insertion of a cassette while gene targeting targets a specific gene. Cassettes can be used for many different things while the flanking homology regions of gene targeting cassettes need to be adapted for each gene. This makes gene trapping more easily amenable for large scale projects than targeting. On the other hand, gene targeting can be used for genes with low transcriptions that would go undetected in a trap screen. Also, the probability of trapping increases with intron size. For gene targeting these compact genes are just as easily altered.

2007 Nobel prize

Mario R. Capecchi, Martin J. Evans and Oliver Smithies were declared laureates of the 2007 Nobel Prize in Physiology or Medicine for their work on "principles for introducing specific gene modifications in mice by the use of embryonic stem cells", or gene targeting.^[1]

References

See also

• genetics, genetic engineering
• gene knockout, knockout mouse
• gene trapping (random gene knockout technique)
• gene knockdown (reduction not removal of a gene product)
• Mus musculus (house mouse; common model organism)
• Physcomitrella patens (gene targetting in plants)
• Toll-like receptor (example of a gene targeted for analysis)

External links

• guide to gene targeting by the University of California, San Diego
• outline of gene targeting by the University of Michigan
• gene targeting diagram & summary by Heydari lab, Wayne State University
• research highlights on reporter genes used in gene targeting