Knockout mouse

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A knockout mouse or knock-out mouse is a genetically modified mouse ( Mus musculus ) where the researcher has disabled, or "knocked out" genes by replacing them or messing them with a piece of artificial DNA. They are important animal models for studying the role of genes that have been sequenced but their function has not been determined. By causing certain genes to become inactive on the mouse, and observing any difference from normal behavior or physiology, the researcher can deduce the possibility of functioning.

The current rat is the closest animal species of labor to which humans can knockout techniques be easily applied. They are widely used in the knockout experiments, especially those who investigate genetic questions related to human physiology. Gene knockout in rats is much more difficult and is only possible since 2003.

The first KO recorded mole was created by Mario R. Capecchi, Martin Evans, and Oliver Smithies in 1989, where they were awarded the 2007 Nobel Prize in Physiology or Medicine. The technological aspect for producing knockout mice, and the mice itself has been patented in many countries by private companies.

Video Knockout mouse

Use

Tapping gene activity provides information about what genes normally do. Humans share many genes with mice. Consequently, observing the characteristics of knockout mice provides researchers with information that can be used to better understand how the same genes can cause or contribute to the disease in humans.

Examples of research where knockout mice have been useful include studying and modeling different types of cancer, obesity, heart disease, diabetes, arthritis, substance abuse, anxiety, aging and Parkinson's disease. Knockout mice also offers a biological and scientific context in which other drugs and therapies can be developed and tested.

Millions of KO mice are used in experiments every year.

Maps Knockout mouse

Strain

There are several thousand different knockout mouse strains. Many mouse models are named after the genes that have been inactivated. For example, the p53 knockout mouse is named after the p53 gene encoding a protein that normally suppresses tumor growth by blocking cell division and/or inducing apoptosis. Humans born with mutations inactivating the p53 gene suffer from Li-Fraumeni syndrome, a condition that dramatically increases the risk of developing bone, breast and blood cancer cancers at an early age. Other mouse models are named according to their physical or behavioral characteristics.

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Procedures

There are several variations in the procedure of producing knockout mice; here is a typical example.

1. The gene to be exposed is isolated from the mouse gene library. Then a new DNA sequence is engineered that is very similar to the original gene and the order of its nearest neighbors, except that it is altered enough to make the gene inoperable. Typically, new sequences are also labeled genes, genes that normal mice do not have and which provide resistance to certain toxic agents (eg, neomycin) or that produce observable changes (eg color or fluorescence). In addition, the second gene, such as herpes tk, is also included in the construct to achieve a complete selection.
2. Embryonic stem cells are isolated from mouse blastocysts (very young embryos) and grow in vitro . For this example, we will take stem cells from white mice.
3. The new sequence of step 1 is inserted into the stem cell from step 2 by electroporation. With the natural process of homologous recombination, some electroporated stem cells will combine new sequences with genes tapped into their chromosomes instead of the original gene. The likelihood of a successful recombination event is relatively low, so that the majority of modified cells will have a new sequence in just one of two relevant chromosomes - they are said to be heterozygous. Vector-altered cells containing neomycin resistance genes and herpes tk genes are grown in a solution containing neomycin and Ganciclovir to choose for the transformation that occurs through homologous recombination. Any DNA insertion that occurs through random insertion will die because they test positive for neomycin resistance genes and herpes tk genes, whose gene products react with Ganciclovir to produce lethal toxins. In addition, cells that do not integrate one of the genetic material test are negative for both genes and therefore die as a result of poisoning with neomycin.
4. Embryonic stem cells that combine genes that are tapped are isolated from unchanged cells using the marker genes from step 1. For example, unchanging cells can be killed by using toxic agents whose cells are turned into resistant./li>
5. Embryonic stem cells that are destroyed from step 4 are inserted into mouse blastocysts. For this example, we use blastocysts from the gray mouse. Blastocysts now contain two types of stem cells: the original (from the gray mouse), and the knocked cell (from the white mouse). The blastocyst is then implanted into the female rat's womb, where they develop. Therefore, the newborn mice will become chimera: some parts of their body are produced from the original stem cell, another part of the stem cell that collapses. Their fur will show white and gray patches, with white patches coming from stem cells that collapse and gray spots from the recipient's blastocyst.
6. Some newborn chimera mice will have gonads derived from stem cells threshed, and will therefore produce eggs or sperm containing the gene exposed. When these chimera mice are crossed with another of the wild type, some of their descendants will have one copy of the gene exposed in all their cells. These mice will be completely white and not chimeras, but they are still heterozygous.
7. When these heterozygous descendants intermarry, some of their descendants will inherit the gene removed from both parents; they do not carry a functional copy of the original gene that does not change (ie they are homozygous for the allele).

A detailed explanation of how a knockout (KO) is made on the Nobel Prize website in Physiology or Medicine 2007.

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Limitations

The National Institutes of Health discusses some important limitations of this technique.

While mouse knockout technology is a valuable research tool, there are some important limitations. About 15 percent of the KO genes are lethal by development, which means that genetically altered embryos can not grow into adult rats. This problem is often overcome through the use of conditional mutations. The lack of adult rats limits the study to embryonic development and often makes it more difficult to determine gene function in relation to human health. In some cases, genes can serve different functions in adults rather than in developing embryos.

Gene tapping may also fail to produce observable changes in mice or may even produce characteristics that are different from those observed in humans where the same gene is inactive. For example, mutations in the p53 gene are associated with more than half of human cancers and often cause tumors in a particular set of tissues. However, when the p53 gene is eliminated in mice, animals develop tumors in different tissues.

There is variability in the overall procedure, depending on the strain from which the stem cells have been lowered. Generally cells derived from strain 129 are used. This specific strain is not suitable for many experiments (eg, behavior), so it is very common to cross breeds to other strains. Some genomic loci have proved very difficult to be disabled. The reason may be the presence of repeated sequences, extensive DNA methylation, or heterochromatin. The confusing presence of about 129 genes in the knockout segment of genetic material has been dubbed the "flanking gene effect". Methods and guidelines for addressing this issue have been proposed.

Another limitation is that conventional knockout rats (ie unconditional) develop without the gene being investigated. Sometimes, the loss of activity during development may mask the role of genes in adulthood, especially if genes are involved in processes that include development. The conditional mutation/induced mutation approach is then required which first allows the mouse to develop and mature normally before the desired gene ablation.

Another serious limitation is the lack of evolutive adaptation in knockout models that may occur in wild-type animals after they naturally mutate. For example, the specific erythrocyte coexpression GLUT1 with stomatin is a compensatory mechanism in mammals that can not synthesize vitamin C.

src: www.pnas.org

See also

• Chimera (genetics)
• Genetically modified organism
• Genetics
• Humouse
• International Knockout Mouse Consortium
• International Phenotyping Mouse Consortium
• Knockout moss
• Oncomouse

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References

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External links

Source of the article : Wikipedia