Knockout mice

A knockout mouse is a genetically engineered mouse that has had one or more of its genes made inoperable through a gene knockout. Knockout is a route to learning about a gene that has been sequenced but has an unknown or incompletely known function. By inactivating the gene and studying the mouse for any resulting differences, researchers can infer the probable function of that gene. Mice are the laboratory animal species most closely related to humans in which the knockout technique can be easily performed, so they are a favorite subject for knockout experiments, especially with regard to genetic questions that relate to human physiology. (Gene knockout in rats is much harder and has only been possible since 2003.) Image File history File links PCWmice1. ... Image File history File links PCWmice1. ... Kenyans examining insect-resistant transgenic Bt corn. ... Binomial name Mus musculus Linnaeus, 1758 Mus musculus is the common house mouse. ... For other uses, see Gene (disambiguation). ... A gene knockout is a genetically engineered organism that carries one or more genes in its chromosomes that has been made inoperative. ... In genetics and biochemistry, sequencing means to determine the primary structure (or primary sequence) of an unbranched biopolymer. ... A model organism is a species that is extensively studied to understand particular biological phenomena, with the expectation that discoveries made in the organism model will provide insight into the workings of other organisms. ... This article is about modern humans. ... Binomial name Rattus norvegicus (Berkenhout, 1769) The Brown Rat or Norway Rat (Rattus norvegicus) is one of the most well-known and common rats, and also one of the largest. ... Year 2003 (MMIII) was a common year starting on Wednesday of the Gregorian calendar. ...

The first knockout mice were produced by Mario Capecchi, Martin Evans and Oliver Smithies in 1987–1989, for which they were awarded the Nobel Prize for Medicine in 2007. Aspects of this technology were licensed to Lexicon Pharmaceuticals. The various methods for generating Knockout mice are extensively patented in the United States. The resulting Knockout mice can also be patented in many countries, including the United States. Mario Capecchi (1937- ) is an Italian-born American molecular geneticist. ... Sir Martin Evans is a British scientist, he is credited with discovering embryonic stem cells in 1981, and for the development of the knockout mouse Categories: Geneticists | Scientist stubs ... Oliver Smithies was awarded a Lasker Award in 2001. ... Year 1987 (MCMLXXXVII) was a common year starting on Thursday (link displays 1987 Gregorian calendar). ... Year 1989 (MCMLXXXIX) was a common year starting on Sunday (link displays 1989 Gregorian calendar). ... The Nobel Prizes (Swedish: ) are awarded for Physics, Chemistry, Literature, Peace, and Physiology or Medicine. ... Year 2007 (MMVII) is the current year, a common year starting on Monday of the Gregorian calendar and the AD/CE era in the. ... Lexicon Genetics Incorporated Lexicon Genetics is a biopharmaceutical company focused on the discovery of breakthrough treatments for human disease. ... For other uses, see Patent (disambiguation). ...

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. Cancer is a class of diseases or disorders characterized by uncontrolled division of cells and the ability of these to spread, either by direct growth into adjacent tissue through invasion, or by implantation into distant sites by metastasis (where cancer cells are transported through the bloodstream or lymphatic system). ... Heart disease is an umbrella term for a number of different diseases which affect the heart and as of 2007 it is the leading cause of death in the United States. ... This article is about the disease that features high blood sugar. ... Arthritis (from Greek arthro-, joint + -itis, inflammation; plural: arthritides) is a group of conditions where there is damage caused to the joints of the body. ... Also see Alcoholism and Drug addiction. ... what up?? Anxiety is a physiological state characterized by cognitive, somatic, emotional, and behavioral components (Seligman, Walker & Rosenhan, 2001). ... Ageing or aging is the process of getting older. ...

Many of these 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. For example, "Methuselah" is a knockout mouse model noted for longevity, while "Frantic" is a model useful for studying anxiety disorders. TP53 bound to a short DNA fragment. ... Li-Fraumeni syndrome is a rare autosomal dominant hereditary disorder. ...

Procedure

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

1. The gene to be knocked out is isolated from a mouse 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 transfers resistance to a certain antibiotic or a selectable marker.
2. From a mouse blastocyst (a very young embryo consisting of a ball of undifferentiated cells with surrounding extraembryonic cells), stem cells are isolated; these can be grown in vitro. For this example, we will take a stem cell from a white mouse.
3. 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 chromosomes 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 antibiotic from step 1, those stem cells that actually did incorporate the new sequence can be quickly isolated from those that did not.
4. The stem cells from step 3 are inserted into mouse blastocyst cells. 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 chimeras: 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.
5. Newborn mice are only useful if the newly engineered sequence was incorporated into the germ cells (egg or sperm cells). So we cross these new mice with others and watch for offspring that are all white. These are then further inbred to produce mice that carry no functional copy of the original gene.

In molecular biology, a library is a collection of molecules in a stable form that represents some aspect of an organism. ... part of a DNA sequence A DNA sequence (sometimes genetic sequence) is a succession of letters representing the primary structure of a real or hypothetical DNA molecule or strand, The possible letters are A, C, G, and T, representing the four nucleotide subunits of a DNA strand (adenine, cytosine, guanine... A marker gene is used in molecular biology to determine if a piece of DNA has been successfully inserted into the host organism. ... Staphylococcus aureus - Antibiotics test plate. ... The blastocyst is an early stage of the human (or any other mammal) development early in pregnancy. ... For other uses, see Embryo (disambiguation). ... Mouse embryonic stem cells with fluorescent marker. ... Wiktionary has a definition of: In vitro In vitro (Latin: within glass) means within a test tube, or, more generally, outside a living organism or cell. ... Electroporation, or electropermeabilization, is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by externally applied electrical field. ... Figure 1: A representation of a condensed eukaryotic chromosome, as seen during cell division. ... Chromosomal crossover is the process by which two chromosomes, paired up during Prophase I of meiosis, exchange some distal portion of their DNA. Crossover occurs when two chromosomes, normally two homologous instances of the same chromosome, break and then reconnect but to the different end piece. ... The blastocyst is an early stage of the human (or any other mammal) development early in pregnancy. ... This article is about female reproductive anatomy. ... It has been suggested that Chimarism be merged into this article or section. ...

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 mutations. 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. For linguistic mutation, see Apophony. ...

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. In plant breeding, backcrossing is a crossing of a hybrid with one of its parents. ... DNA methylation is a type of chemical modification of DNA that can be inherited without changing the DNA sequence. ... For differently-colored eyes, see Heterochromia. ...

See also

• Transgenic animal
• Genetics
• Gene knockout
• Mus musculus (house mouse)
• Humouse
• Lexicon genetics

Transgenic animals are animals produced with externally introduced genes. ... This article is about the general scientific term. ... A gene knockout is a genetically engineered organism that carries one or more genes in its chromosomes that has been made inoperative. ... Binomial name Mus musculus Linnaeus, 1758 Mus musculus is the common house mouse. ... A humouse is an immunodeficient mouse reconstituted with a human immune system. ... Lexicon Genetics Incorporated Lexicon Genetics is a biopharmaceutical company focused on the discovery of breakthrough treatments for human disease. ...

References

• NHGRI. 2006. Knockout Mice

External links

• Texas Institute for Genomic Medicine
• Knockout Mouse Bioinformatics, Protocols and Services
• Studying Gene Function: Creating Knockout Mice - a review from the Science Creative Quarterly
• - The Knock Out Mouse Project (KOMP) website
• Disease - Transgenic, Knockout Mouses - BSRC Alexander Fleming
• Transgenic, Knockout Mouses - Biomedcode