Knockout genetics and knockout mice
Knockout Methodology (Genetics & Inherited Conditions)
Before knockout mice, transgenic animals had been generated in which “foreign” DNA was incorporated into their genomes in a largely haphazard fashion; such animals should more properly be referred to as “genetically modified.” In contrast, knockout technology targets a particular gene to be altered. Prior to the creation of transgenic animals, any genetic change resulted from spontaneous and largely random mutations. Individual variability and inherited diseases are the results of this natural phenomenon—as are, on a longer time frame, the evolutionary changes responsible for the variety of living species on the earth. Spontaneously generated animal models of human inherited diseases have been helpful in understanding mutations and developing treatments for them. However, these mutants were essentially gifts of nature, and their discovery was largely serendipitous. In knockout mice, animal models are directly generated, expediting study of the pathology and treatment of inherited diseases.
In a knockout mouse, a single gene is selected to be inactivated in such a way that the nonfunctional gene is reliably passed to its progeny. Developed independently by Mario Capecchi at the University of Utah and Oliver Smithies of the University of North Carolina, the process is formally termed “targeted gene inactivation,” and, although simple in concept, it is operationally complex and technically demanding. It...
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Usefulness of Knockout Mice (Genetics & Inherited Conditions)
Knockout mice are important because they permit the function of a specific gene to be established, and, since mice and humans share 99 percent of the same genes, the results can often be applied to people. However, knockout mice are not perfect models, in that some genes are specific to mice or humans, and similar genes can be expressed at different levels in the two species. Nevertheless, knockout mice are vastly superior to spontaneous mutants because the investigator selects the gene to be modified. Mice are predominantly used in this technology because of their short generation interval and small size; the short generation interval accelerates the breeding program necessary to establish pure strains, and the small size reduces the space and food needed to house and sustain them.
Knockout mice are, first of all, excellent animal models for inherited diseases, the study of which was the initial impetus for their creation. The Lesch-Nyhan syndrome, a neurological disorder, was the focus of much of the early work with the knockout technology. The methodology has permitted the creation of previously unknown animal models for cystic fibrosis, Alzheimer’s disease, and sickle-cell disease, which will stimulate research into new therapies for these diseases. Knockout mice have also been developed to study atherosclerosis, cancer susceptibility, and obesity, as well as immunity, memory, learning, behavior, and...
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Double Knockouts, Conditional Knockouts, and Reverse Knockouts (Genetics & Inherited Conditions)
Redundancy is fairly common in gene function: Often, more than one gene has responsibility for the same or similar activity in vivo. Eliminating one redundant gene may have little consequence because another gene can fulfill its function. This has led to the creation of double knockout mice, in which two specific genes are eliminated. Double knockouts are generated by crossing two separate single knockout mice to produce double mutant offspring. Consequences of both mutations can then be examined simultaneously.
Some single knockout mice are deleteriously affected during embryonic development and do not survive to birth. This has led to the generation of conditional knockout mice, in which the gene is functional until a particular stage of life or tissue development triggers its inactivation. The approach is to generate animals with two mutations: The first is the addition of a new gene that causes a marked segment of a gene to be deleted in response to a temporal or tissue signal, and the second is to mark the gene that has been selected to be excised. In these animals, the latter gene remains functional until signaled to be removed.
Knockout methodology involves generation of loss-of-function or null mutations. Its reversal would permit the function of an inoperative gene to be restored. This reversal has been successfully accomplished in mice with the correction of the...
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Further Reading (Genetics & Inherited Conditions)
Capecchi, Mario. “Targeted Gene Replacement.” Scientific American 270, no. 3 (March, 1994): 52. One of the originators of the technology describes the steps involved and examples of its utility.
Crawley, Jacqueline N. What’s Wrong with My Mouse? Behavioral Phenotyping of Transgenic and Knockout Mice. 2d ed. Hoboken, N.J.: Wiley-Interscience, 2007. Discusses transgenic technology and the mouse genome. Illustrations, bibliography, and index.
Gilbert, Scott F. Developmental Biology. 8th ed. Sunderland, Mass.: Sinauer Associates, 2006. Includes a discussion of knockout methodology. Bibliography.
Kühn, Ralf, and Wolfgang Wurst, eds. Gene Knockout Protocols. 2d ed. New York: Humana Press, 2009. Collection of laboratory protocols describing the mice mutagenesis techniques developed since 2002, including stem cell manipulation, the generation of genetically engineered mice, and mutant phenotype analysis. Designed for researchers and scientists.
Mak, Tak W., et al., eds. The Gene Knockout Factsbook. 2 vols. San Diego: Academic Press, 1998. Covers six hundred gene knockouts, including their general descriptions, constructs, and phenotypes. Bibliography, index.
Mestel, Rosie. “The Mice Without Qualities.” Discover 14, no. 3 (March, 1993): 18. Briefly reports on the creation of knockout mice and their use in understanding the role of the...
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Web Site of Interest (Genetics & Inherited Conditions)
International Knockout Mouse Consortium. http://www.knockoutmouse.org. Consortium members are working together to mutate all protein-coding genes in the mouse using a combination of gene trapping and gene targeting in embryonic stem cells. The group’s Web site contains a database enabling users to retrieve information about specific mouse genes and chromosomes, as well as other information about mice genetics.
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