Gene targeting

Gene targeting (also, replacement strategy based on homologous recombination) is a genetic technique that uses homologous recombination to modify an endogenous gene. The method can be used to delete a gene, remove exons, add a gene and modify individual base pairs (introduce point mutations). The process of gene targeting provides a way to alter specific genes in order to better identify their biological roles.[1] 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.

A chimeric mouse gene targeted for the agouti coat color gene, with its offspring


In general, DNA containing part of the gene to be targeted, a reporter gene, and a (dominant) selectable marker is assembled in bacteria.

Gene targeting methods are established for several model organisms and may vary depending on the species used. To target genes in mice, the DNA is inserted into mouse embryonic stem cells in culture. Cells with the insertion can contribute to a mouse's tissue via embryo injection. Finally, chimeric mice where the modified cells make up the reproductive organs are bred. After this step the entire body of the mouse is based on the selected embryonic stem cell.

Wild-type Physcomitrella and knockout-mosses: Deviating phenotypes induced in gene-disruption library transformants. Physcomitrella wild-type and transformed plants were grown on minimal Knop medium to induce differentiation and development of gametophores. For each plant, an overview (upper row, scale bar corresponds to 1 mm) and a close-up (bottom row, scale bar equals 0.5 mm) is shown. A, Haploid wild-type moss plant completely covered with leafy gametophores and close-up of wild-type leaf. B-D, Different Mutants.[2]

To target genes in moss, the DNA is incubated together with freshly isolated protoplasts and with polyethylene glycol. As mosses are haploid organisms,[3] moss filaments (protonema) can be directly screened for the target, either by treatment with antibiotics or with PCR. Unique among plants, this procedure for reverse genetics is as efficient as in yeast.[4] Gene targeting has 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,[5] engineered homing endonucleases,[6] and nucleases based on engineered TAL effectors.[7] This method has been applied to species including Drosophila melanogaster,[5] tobacco,[8][9] corn,[10] human cells,[11] mice[12] and rats.[12]

Comparison with gene trapping

Gene trapping is based on random insertion of a cassette, while gene targeting manipulates 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. The probability of trapping increases with intron size, while for gene targeting, small genes are just as easily altered.


Gene targeting has been widely used to study human genetic diseases by removing ("knocking out"), or adding ("knocking in"), specific mutations of interest.[13] Previously used to engineer rat cell models, advances in gene targeting technologies enable a new wave of isogenic human disease models. These models are the most accurate in vitro models available to researchers and facilitate the development of personalized drugs and diagnostics, particularly in oncology.[14]

2007 Nobel Prize

Mario R. Capecchi, Martin J. Evans and Oliver Smithies were awarded 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.[15]

See also


  1. Hall B, Limaye A, Kulkarni AB (September 2009). "Overview: generation of gene knockout mice". Current Protocols in Cell Biology. 19 (1): Unit 19.12 19.12.1-Unit 19.12 19.1217. doi:10.1002/0471143030.cb1912s44. PMC 2782548. PMID 19731224.
  2. Egener T, Granado J, Guitton MC, Hohe A, Holtorf H, Lucht JM, et al. (July 2002). "High frequency of phenotypic deviations in Physcomitrella patens plants transformed with a gene-disruption library". BMC Plant Biology. 2: 6. doi:10.1186/1471-2229-2-6. PMC 117800. PMID 12123528.
  3. Ralf Reski (1998): Development, genetics and molecular biology of mosses. Botanica Acta 111, 1-15.
  4. Ralf Reski(1998): Physcomitrella and Arabidopsis: the David and Goliath of reverse genetics. Trends Plant in Science 3, 209-210.
  5. Bibikova M, Beumer K, Trautman JK, Carroll D (May 2003). "Enhancing gene targeting with designed zinc finger nucleases". Science. 300 (5620): 764. doi:10.1126/science.1079512. PMID 12730594. S2CID 42087531.
  6. Grizot S, Smith J, Daboussi F, Prieto J, Redondo P, Merino N, et al. (September 2009). "Efficient targeting of a SCID gene by an engineered single-chain homing endonuclease". Nucleic Acids Research. 37 (16): 5405–5419. doi:10.1093/nar/gkp548. PMC 2760784. PMID 19584299.
  7. Miller JC, Tan S, Qiao G, Barlow KA, Wang J, Xia DF, et al. (February 2011). "A TALE nuclease architecture for efficient genome editing". Nature Biotechnology. 29 (2): 143–148. doi:10.1038/nbt.1755. PMID 21179091. S2CID 53549397.
  8. Cai CQ, Doyon Y, Ainley WM, Miller JC, Dekelver RC, Moehle EA, et al. (April 2009). "Targeted transgene integration in plant cells using designed zinc finger nucleases". Plant Molecular Biology. 69 (6): 699–709. doi:10.1007/s11103-008-9449-7. PMID 19112554. S2CID 6826269.
  9. Townsend JA, Wright DA, Winfrey RJ, Fu F, Maeder ML, Joung JK, Voytas DF (May 2009). "High-frequency modification of plant genes using engineered zinc-finger nucleases". Nature. 459 (7245): 442–445. Bibcode:2009Natur.459..442T. doi:10.1038/nature07845. PMC 2743854. PMID 19404258.
  10. Shukla VK, Doyon Y, Miller JC, DeKelver RC, Moehle EA, Worden SE, et al. (May 2009). "Precise genome modification in the crop species Zea mays using zinc-finger nucleases". Nature. 459 (7245): 437–441. Bibcode:2009Natur.459..437S. doi:10.1038/nature07992. PMID 19404259. S2CID 4323298.
  11. Urnov FD, Miller JC, Lee YL, Beausejour CM, Rock JM, Augustus S, et al. (June 2005). "Highly efficient endogenous human gene correction using designed zinc-finger nucleases". Nature. 435 (7042): 646–651. Bibcode:2005Natur.435..646U. doi:10.1038/nature03556. PMID 15806097. S2CID 4390010.
  12. Cui X, Ji D, Fisher DA, Wu Y, Briner DM, Weinstein EJ (January 2011). "Targeted integration in rat and mouse embryos with zinc-finger nucleases". Nature Biotechnology. 29 (1): 64–67. doi:10.1038/nbt.1731. PMID 21151125. S2CID 13409267.
  13. Fanelli A (2017). "Applications of Xenografting". Retrieved 15 January 2018.
  14. Sur S, Pagliarini R, Bunz F, Rago C, Diaz LA, Kinzler KW, et al. (March 2009). "A panel of isogenic human cancer cells suggests a therapeutic approach for cancers with inactivated p53". Proceedings of the National Academy of Sciences of the United States of America. 106 (10): 3964–3969. Bibcode:2009PNAS..106.3964S. doi:10.1073/pnas.0813333106. PMC 2656188. PMID 19225112.
  15. "Press Release: The 2007 Nobel Prize in Physiology or Medicine". Retrieved 2007-10-08.
  16. Bouché N, Bouchez D (April 1, 2001). "Arabidopsis gene knockout: phenotypes wanted". Current Opinion in Plant Biology. 4 (2): 111–117. doi:10.1016/S1369-5266(00)00145-X. PMID 11228432 via ScienceDirect.
This article is issued from Wikipedia. The text is licensed under Creative Commons - Attribution - Sharealike. Additional terms may apply for the media files.