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, add a gene, 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.
Contents
Methods[edit]
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,[2] 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.[3] 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,[4] engineered homing endonucleases,[5] and nucleases based on engineered TAL effectors.[6] To date, this method has been applied to a number of species including Drosophila melanogaster,[4] tobacco,[7][8] corn,[9] human cells,[10] mice,[11] and rats.[11]
Comparison with gene trapping[edit]
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.
Applications[edit]
Gene targeting has been widely used to study human genetic diseases by removing ("knocking out"), or adding ("knocking in"), specific mutations of interest to a variety of models. Previously used to engineer rat cell models, advances in gene targeting technologies are enabling the creation of a new wave of isogenic human disease models. These models are the most accurate in-vitro models available to researchers to date, and are facilitating the development of new personalized drugs and diagnostics, particularly in the field of cancer.[12]
2007 Nobel prize[edit]
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.[13]
See also[edit]
- Cre recombinase
- Cre-Lox recombination
- FLP-FRT recombination
- Gene trapping (random gene knockout technique)
- Genetic recombination
- Homologous recombination
- Recombinase-mediated cassette exchange (exchange of a preexisting "gene cassette" for an "gene of interest")
- Site-specific recombinase technology
- Toll-like receptor (example of a gene targeted for analysis)
- Mus musculus (house mouse; common model organism)
- Physcomitrella patens (only plant in which gene targeting is available, as of 1998[14])
References[edit]
- ^ Egener, T.; Granado, J.; Guitton, M. C.; Hohe, A.; Holtorf, H.; Lucht, J. M.; Rensing, S. A.; Schlink, K.; Schulte, J.; Schween, G.; Zimmermann, S.; Duwenig, E.; Rak, B.; Reski, R. (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.
- ^ Ralf Reski (1998): Development, genetics and molecular biology of mosses. Botanica Acta 111, 1-15.
- ^ Ralf Reski(1998): Physcomitrella and Arabidopsis: the David and Goliath of reverse genetics. Trends Plant in Science 3, 209-210. [1]
- ^ a b Bibikova, M.; Beumer, K.; Trautman, J.; Carroll, D. (2003). "Enhancing Gene Targeting with Designed Zinc Finger Nucleases". Science. 300 (5620): 764. doi:10.1126/science.1079512. PMID 12730594.
- ^ Grizot, S.; Smith, J.; Daboussi, F.; Prieto, J.; Redondo, P.; Merino, N.; Villate, M.; Thomas, S.; Lemaire, L.; Montoya, G.; Blanco, F. J.; Pâques, F.; Duchateau, P. (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.
- ^ Miller, J. C.; Tan, S.; Qiao, G.; Barlow, K. A.; Wang, J.; Xia, D. F.; Meng, X.; Paschon, D. E.; Leung, E.; Hinkley, S. J.; Dulay, G. P.; Hua, K. L.; Ankoudinova, I.; Cost, G. J.; Urnov, F. D.; Zhang, H. S.; Holmes, M. C.; Zhang, L.; Gregory, P. D.; Rebar, E. J. (2010). "A TALE nuclease architecture for efficient genome editing". Nature Biotechnology. 29 (2): 143–148. doi:10.1038/nbt.1755. PMID 21179091.
- ^ Cai, C. Q.; Doyon, Y.; Ainley, W. M.; Miller, J. C.; Dekelver, R. C.; Moehle, E. A.; Rock, J. M.; Lee, Y. L.; Garrison, R.; Schulenberg, L.; Blue, R.; Worden, A.; Baker, L.; Faraji, F.; Zhang, L.; Holmes, M. C.; Rebar, E. J.; Collingwood, T. N.; Rubin-Wilson, B.; Gregory, P. D.; Urnov, F. D.; Petolino, J. F. (2008). "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. ISSN 0167-4412. PMID 19112554.
- ^ Townsend, J. A.; Wright, D. A.; Winfrey, R. J.; Fu, F.; Maeder, M. L.; Joung, J. K.; Voytas, D. F. (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.
- ^ Shukla, V. K.; Doyon, Y.; Miller, J. C.; Dekelver, R. C.; Moehle, E. A.; Worden, S. E.; Mitchell, J. C.; Arnold, N. L.; Gopalan, S.; Meng, X.; Choi, V. M.; Rock, J. M.; Wu, Y. Y.; Katibah, G. E.; Zhifang, G.; McCaskill, D.; Simpson, M. A.; Blakeslee, B.; Greenwalt, S. A.; Butler, H. J.; Hinkley, S. J.; Zhang, L.; Rebar, E. J.; Gregory, P. D.; Urnov, F. D. (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.
- ^ Urnov, F. D.; Miller, J. C.; Lee, Y. L.; Beausejour, C. M.; Rock, J. M.; Augustus, S.; Jamieson, A. C.; Porteus, M. H.; Gregory, P. D.; Holmes, M. C. (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.
- ^ a b Cui, X.; Ji, D.; Fisher, D. A.; Wu, Y.; Briner, D. M.; Weinstein, E. J. (2010). "Targeted integration in rat and mouse embryos with zinc-finger nucleases". Nature Biotechnology. 29 (1): 64–7. doi:10.1038/nbt.1731. PMID 21151125.
- ^ A Panel of Isogenic Human Cancer Cells Suggests a Therapeutic Approach for Cancers with Inactivated p53 Proc Natl Acad Sci U S A Printed online at www.pnas.org/cgi/doi/10.1073/pnas.0813333106
- ^ "Press Release: The 2007 Nobel Prize in Physiology or Medicine". Retrieved 2007-10-08.
- ^ Arabidopsis gene knockout: phenotypes wanted
External links[edit]
- Guide to gene targeting by the University of California, San Diego
- Outline of gene targeting by the University of Michigan
- Gene targeting in mouse diagram & summary by Heydari lab, Wayne State University
- Research highlights on reporter genes used in gene targeting
- Targeted gene replacement in barley