Targeted gene replacement: Difference between revisions
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'''Targeted gene replacement''' is a technique in which a [[cloning|cloned]] piece of [[DNA]] with a [[gene]] of interest is first modified in vitro, typically by intragenic (i.e. within the gene) and exogenic (outside of the gene) insertions of [[antibiotic]] or [[virus|viral]] resistance genes. The cloned mutant DNA is then introduced into [[mammal]]ian [[stem cell]]s in culture. | '''Targeted gene replacement''' is a technique in which a [[cloning|cloned]] piece of [[DNA]] with a [[gene]] of interest is first modified [[in vitro]], typically by intragenic (i.e. within the gene) and exogenic (outside of the gene) insertions of [[antibiotic]] or [[virus|viral]] resistance genes. The cloned mutant DNA is then introduced into [[mammal]]ian [[stem cell]]s in culture. | ||
==Summary== | ==Summary== | ||
In [[prokaryote]]s and simple [[eukaryote]]s, the effects of [[mutation]]s can be determined by isolating spontaneous mutations in colony populations, or by inducing mutations with [[DNA]]-damaging | In [[prokaryote]]s and simple [[eukaryote]]s, the effects of [[mutation]]s can be determined by isolating spontaneous mutations in colony populations, or by inducing mutations with [[DNA]]-damaging mutagens. Neither method is useful for producing a mutation in a specific [[genome|genomic]] location, nor are the techniques applicable to less simple and prolific organisms like [[mammal]]s. | ||
Targeted gene replacement was introduced in the 1980s to surmount these difficulties and allow specific [[mutation]]s to be induced in [[mammal]]ian cells. Even a mammalian [[somatic]] [[cell]], it turns out, has the ability to integrate [[DNA]] fragments into its [[genome]] via [[homologous]] [[recombination]]. In some cases, the new, disrupted gene will "target" and replace its corresponding non-mutant gene on one strand of the genome. In these instances, only the | Targeted gene replacement was introduced in the 1980s to surmount these difficulties and allow specific [[mutation]]s to be induced in [[mammal]]ian cells. Even a mammalian [[somatic]] [[cell]], it turns out, has the ability to integrate [[DNA]] fragments into its [[genome]] via [[homologous]] [[recombination]]. In some cases, the new, disrupted gene will "target" and replace its corresponding non-mutant gene on one strand of the genome. In these instances, only the intragenic gene will be incorporated into the host DNA, and the cells in which this targeted gene replacement occur will possess only the intragenic resistance — allowing them to be isolated. These recombinant [[stem cell]]s may then be introduced into a developing [[embryo]], where they will divide like other cells and eventually give rise to whole [[tissue]]s in the organism. Any [[germ-line]] mutants that result can be further interbred to produce [[homozygote]]s in which the gene is only present as the disrupted [[allele]], and the effects of the non-expression of the gene may determined by comparison with normal individuals. | ||
==Related publications== | ==Related publications== | ||
Latest revision as of 00:44, 8 May 2008
Targeted gene replacement is a technique in which a cloned piece of DNA with a gene of interest is first modified in vitro, typically by intragenic (i.e. within the gene) and exogenic (outside of the gene) insertions of antibiotic or viral resistance genes. The cloned mutant DNA is then introduced into mammalian stem cells in culture.
Summary
In prokaryotes and simple eukaryotes, the effects of mutations can be determined by isolating spontaneous mutations in colony populations, or by inducing mutations with DNA-damaging mutagens. Neither method is useful for producing a mutation in a specific genomic location, nor are the techniques applicable to less simple and prolific organisms like mammals.
Targeted gene replacement was introduced in the 1980s to surmount these difficulties and allow specific mutations to be induced in mammalian cells. Even a mammalian somatic cell, it turns out, has the ability to integrate DNA fragments into its genome via homologous recombination. In some cases, the new, disrupted gene will "target" and replace its corresponding non-mutant gene on one strand of the genome. In these instances, only the intragenic gene will be incorporated into the host DNA, and the cells in which this targeted gene replacement occur will possess only the intragenic resistance — allowing them to be isolated. These recombinant stem cells may then be introduced into a developing embryo, where they will divide like other cells and eventually give rise to whole tissues in the organism. Any germ-line mutants that result can be further interbred to produce homozygotes in which the gene is only present as the disrupted allele, and the effects of the non-expression of the gene may determined by comparison with normal individuals.
Related publications
- Capecci MR (1994) Targeted gene replacement. Scientific American (March): 52-59.
- Griffiths AJF, Miller JH, Suzuki DT, Lewontin RC, Gelbart WM (1996) An introduction to genetic analysis (6th ed). New York: W. H. Freeman and Company, ch. 15.
- Tang YP, Shimizu E, Tsien JZ (1999) Genetic enhancement of learning and memory in mice. Nature 401(6748): 63.