Knockout mouse 340919 222379396 2008-06-29T01:19:24Z Crusio 3258996 rvt: Methuselah Prize stipulates *any* method, not just KO (could be nutrition, classical genetics, etc) [[Image:PCWmice1.jpg|thumb|Knockout mice]] A '''knockout mouse''' is a [[genetic engineering|genetically engineered]] [[mus musculus|mouse]] in which one or more [[gene]]s have been turned off through a [[gene knockout]]. Knockout mice are important [[animal model|animal models]] for studying the role of genes which have been [[sequencing|sequenced]], but have unknown functions. By causing a specific gene to be inactive in the mouse, and observing any differences from normal behaviour or condition, researchers can infer its probable function. Mice are currently the most closely related [[model organism|laboratory animal species]] to [[human]]s, for which the knockout technique can easily be applied. They are widely used in knockout experiments, especially those investigating genetic questions that relate to human physiology. Gene knockout in [[rattus norvegicus|rats]] is much harder{{Fact|date=December 2007}} and has only been possible since [[2003]]. The first knockout mouse was created by [[Mario R. Capecchi]], [[Martin Evans]] and [[Oliver Smithies]] in [[1989]], for which they were awarded the [[Nobel Prize]] for Medicine in [[2007]]. Aspects of the technology for generating Knockout mice, and the mice themselves have been [[patent|patented]] in many countries by private companies. ==Use== Knocking out the activity of a gene provides information about what that gene normally does. Humans share many genes with mice. Consequently, observing the characteristics of knockout mice gives researchers information that can be used to better understand how a similar gene may cause or contribute to disease in humans. Examples of research in which knockout mice have been useful include studying and modeling different kinds of [[cancer]], [[obesity]], [[heart disease]], [[diabetes]], [[arthritis]], [[substance abuse]], [[anxiety]], [[aging]] and [[Parkinson's disease]]. Knockout mice also offer a biological and scientific context in which drugs and other therapies can be developed and tested. Millions of knockout mice are used in experiments each year.<ref>[http://www.genome.gov/10005834 genome.gov | Background on Mouse as a Model Organism<!-- Bot generated title -->]</ref> ==Strains== There are several thousand different strains of knockout mice.<ref>[http://www.genome.gov/10005834 genome.gov | Background on Mouse as a Model Organism<!-- Bot generated title -->]</ref> Many of mouse models are named after the gene that has been inactivated. For example, the [[p53]] knockout mouse is named after the p53 gene which codes for a protein that normally suppresses the growth of tumors by arresting cell division. Humans born with mutations that inactivate the p53 gene suffer from [[Li-Fraumeni syndrome]], a condition that dramatically increases the risk of developing bone cancers, breast cancer and blood cancers at an early age. Other mouse models are named, often with creative flair, according to their physical characteristics or behaviors. ==Procedure== [[Image:Knockout mouse production 2.svg|thumb|right|300px|The procedure for making mixed-gentotype blastocyst.]] [[Image:Knockout_mouse_breeding_scheme.svg|thumb|right|300px|Breeding scheme for producing knockout mice. Blastocysts containing cells, that are both wildtype and knockout cells, are injected into the [[uterus]] of a foster mother. This produces offspring that are either wildtype and colored the same color as the blastocyst donor (grey) or chimera (mixed) and partially knocked out. The chimera mice are crossed with a normal wildtype mouse(grey). This produces offspring that are either white and heterozygous for the knocked out gene or grey and wildtype. White heterozygous mice can subsequently be crossed to produce mice that are homozygous for the knocked out gene.]] There are several variations to the procedure of producing knockout mice; the following is a typical example. #The gene to be knocked out is isolated from a mouse [[library (biology)|gene library]]. Then a new [[DNA sequence]] is engineered which is very similar to the original gene and its immediate neighbor sequence, except that it is changed sufficiently to make it inoperable. Usually, the new sequence is also given a [[marker gene]], a gene that normal mice don't have and that confers resistance to a certain toxic agent or that produces an observable change (e.g. colour or fluorescence). The chances of a successful recombination event are relatively low, so the majority of altered cells will have the gene changed for only one of the two relevant chromosomes - they are said to be [[heterozygous]]. #From a mouse [[blastocyst]] (a very young [[embryo]] consisting of a ball of undifferentiated cells with surrounding extraembryonic cells), [[stem cell]]s are isolated; these can be grown ''[[in vitro]]''. For this example, we will take a stem cell from a white mouse. #The stem cells from step 2 are combined with the new sequence from step 1. This is done via [[electroporation]] (using electricity to transfer the DNA across the cell membrane). Some of the electroporated stem cells will incorporate the new sequence into their [[chromosome]]s in place of the old gene; this is called [[homologous recombination]]. The reason for this process is that the new and the old sequence are very similar. Using the marker gene from step 1, those stem cells that actually did incorporate the new sequence can be quickly isolated from those that did not. #The stem cells from step 3 are inserted into a mouse [[blastocyst]]. For this example, we use blastocysts from a grey mouse. These blastocysts are then implanted into the [[uterus]] of female mice, to complete the pregnancy. The blastocysts contain two types of stem cells: the original ones (grey mouse), and the newly engineered ones (white mouse). The newborn mice will therefore be [[Chimera (genetics)#Chimeras in research|chimera]]s: parts of their bodies result from the original stem cells, other parts result from the engineered stem cells. Their furs will show patches of white and grey. #Newborn mice are only useful if the newly engineered sequence was incorporated into the germ cells (egg or sperm cells). These new mice are crossed with others of the white type for offspring that are all white. These mice still contain one functional copy of the gene and must be further inbred to produce mice that carry no functional copy of the original gene (i.e. are [[homozygous]] for that allele). A detailed explanation of how knockout (KO) mice are created is located at the website of The Nobel Prize in Physiology or Medicine 2007 <ref>[http://nobelprize.org/nobel_prizes/medicine/laureates/2007/adv.html The Nobel Prize in Physiology or Medicine 2007]</ref>. ==Limitations== While knockout mice technology represents a valuable research tool, some important limitations exist. About 15 percent of gene knockouts are developmentally lethal, which means that the genetically altered embryos cannot grow into adult mice. This problem is often overcome through the use of [[conditional mutation]]s. The lack of adult mice limits studies to embryonic development and often makes it more difficult to determine a gene's function in relation to human health. In some instances, the gene may serve a different function in adults than in developing embryos. Knocking out a gene also may fail to produce an observable change in a mouse or may even produce different characteristics from those observed in humans in which the same gene is inactivated. For example, mutations in the p53 gene are associated with more than half of human cancers and often lead to tumors in a particular set of tissues. However, when the p53 gene is knocked out in mice, the animals develop tumors in a different array of tissues. There is variability in the whole procedure depending largely on the strain from which the stem cells have been derived. Generally cells derived from strain '''129''' are used. This specific strain is not suitable for many experiments (e.g., behavioral), so it is very common to [[backcross]] the offspring to other strains. Some genomic loci have been proven very difficult to knock out. Reasons might be the presence of repetitive sequences, extensive [[DNA methylation]], or [[heterochromatin]]. == See also == * [[Transgenic animal]] * [[Genetics]] * [[Gene knockout]] * [[Mus musculus]] (house mouse) * [[Humouse]] * [[Lexicon genetics]] ==References== {{Reflist|1}} == External links == * [http://www.scq.ubc.ca/?p=264 Studying Gene Function: Creating Knockout Mice] - a review from the Science Creative Quarterly * [http://www.komp.org The Knock Out Mouse Project (KOMP) website] * [http://www.informatics.jax.org Mouse Genome Informatics (MGI) website] - community model organism database for the laboratory mouse [[Category:Genetically modified organisms]] [[Category:Mice]] [[de:Knockout-Maus]] [[es:Ratón Knockout]] [[fr:Souris knock-out]] [[it:Topo knockout]] [[sv:Knockutmöss]] [[zh:基因剔除小鼠]]