The Knockout mouse.

A laboratory mouse in which a gene affecting hair growth has been knocked out (left), is shown next to a normal lab mouse.

A knockout mouse is a genetically engineered mouse in which one or more genes have been turned off through a targeted mutation.
Knockout mice are important animal models for studying the role of genes which have been sequenced but whose functions have not been determined. 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 laboratory animal species to humans 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 rats is much harder 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 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.

Strains

A knockout mouse (left) that is a model of obesity, compared with a normal mouse.

There are several thousand different strains of knockout mice. Many 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 tumours by arresting cell division. Humans born with mutations that deactivate 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 behaviours.

Procedure

The procedure for making mixed-gentotype blastocyst.

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 neighbour 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.
2. From a mouse blastocyst (a very young embryo consisting of a ball of undifferentiated cells with surrounding extra-embryonic 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 sequences 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.
4. 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 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). 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.

Limitations

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 coloured the same colour 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.

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

While knockout mouse 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.

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 tumours in a particular set of tissues. However, when the p53 gene is knocked out in mice, the animals develop tumours 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., behavioural), 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. The confounding presence of neighbouring 129 genes on the knockout segment of genetic material has been dubbed the "flanking-gene effect". Methods and guidelines to deal with this problem have been proposed.

Another limitation is that conventional (i.e. non-conditional) knockout mice develop in the absence of the gene being investigated. At times, loss of activity during development may mask the role of the gene in the adult state, especially if the gene is involved in numerous processes spanning development. Conditional/inducible mutation approaches are then required that first allow the mouse to develop and mature normally prior to ablation of the gene of interest.