A Knockout Mouse is a genetically engineered mouse in which researchers have inactivated, or “knocked out,” an existing gene by replacing it or disrupting it with an artificial piece of DNA. The loss of gene activity frequently causes changes in a mouse’s phenotype, which includes appearance, behavior, or other apparent and biochemical characteristics.
Knockout mice are significant animal models for studying the role of genes which have been sequenced but whose functions haven’t been established. By causing a precise gene to be inactive in the mouse, and monitoring any differences from normal behavior or physiology, researchers can assume its possible function.
Mice are currently the most closely related laboratory animal species to humans for which the knockout method can easily be applied. They are widely used in knockout experiments, particularly those investigating genetic questions that relate to human physiology. Gene knockout within rats is much more difficult and has only been possible since 2003.
The first documented knockout mouse was created by Mario R. Capecchi, Martin Evans, and Oliver Smithies in 1989, for which they were rewarded the 2007 Nobel Prize in Physiology or Medicine. Aspects of the technology for producing knockout mice, and the mice themselves, have been patented in many countries by private companies.
Knocking out the activity of a gene supplies information about what that gene usually does. Humans have many of the same genes as mice. As a result, observing the characteristics of knockout mice gives researchers information that can be utilized to better understand how a similar gene might cause or contribute to disease in humans.
Examples of research in which knockout mice have proved to be useful include studying and modeling different kinds of obesity, cancer, heart disease, arthritis, diabetes, substance abuse, aging, anxiety, and Parkinson’s disease. Knockout mice also present a biological and scientific context in which drugs and other therapies can be developed and tested.
There are several thousand different strains of these knockout mice. A lot of mouse models are named after the gene that has been inactivated. For instance, the p53 knockout mouse is named after the p53 gene which codes for a protein that usually suppresses the growth of tumors by arresting cell division and/or inducing apoptosis. Humans that are born with mutations that deactivate the p53 gene suffer from Li-Fraumeni syndrome, a condition that significantly increases the risk of developing bone cancers, breast cancer, and blood cancers at an early age. Other mouse models are named, frequently with creative flair, according to their physical characteristics or behaviors.
There are several variations regarding the procedure of producing knockout mice.
Step one, the gene to be knocked out is separated from a mouse gene library. Then a new DNA sequence is engineered which is much like that of the original gene and its immediate neighbor sequence, except that it’s changed sufficiently to make the gene inoperable. Normally, the new sequence is also given a marker gene, a gene that normal mice do not have and that confers resistance to a certain toxic agent or that produces a noticeable change. Step two, stem cells are isolated from a mouse blastocyst and grown in vitro. Step three, the new sequence from step one is introduced into the stem cells from step two via electroporation. By the natural process of homologous recombination, some of the electroporated stem cells will incorporate the new sequence with the knocked-out gene into their chromosomes in place of the original gene. The chances of a successful recombination event are comparatively low, so the majority of altered cells will have the new sequence in only one of the two relevant chromosomes – they are said to be heterozygous. Step four, the stem cells that incorporated the knocked-out gene are isolated from the unaltered cells utilizing the marker gene from step one. Step five, the knocked-out stem cells from step four are inserted into a mouse blastocyst. The blastocysts now contain two types of stem cells: the original ones, and the knocked-out cells. These blastocysts are then implanted into the uterus of a female mouse, where they develop. The newborn mice will therefore be chimeras: some parts of their bodies result from the original stem cells, other parts from the knocked out ones. Step six, some of the newborn chimera mice will have gonads derived from knocked out stem cells, and will therefore produce eggs or sperm containing the knocked out gene. When these chimera mice are crossbred with others of the wild type, some of their offspring will have one copy of the knocked out gene in all their cells. Step seven, when these heterozygous offspring are interbred, some of their offspring will inherit the knocked out gene from both parents; they carry no functional copy of the original unaltered gene.
Although 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 can’t grow into adult mice. This issue is frequently overcome through the usage of conditional mutations. The lack of adult mice limits studies to embryonic development and often makes it harder to determine a gene’s function in relation to human health. In some cases, the gene might serve a different function in adults than in developing embryos.
Knocking out a gene might also fail to produce a noticeable change in a mouse or might even produce different characteristics from those observed in humans in which the same gene is inactivated.
There is variability in the entire procedure depending largely on the strain from which the stem cells have been derived. Usually, cells derived from strain 129 are used. This specific strain isn’t suitable for many experiments, so it’s very common to backcross the offspring to other strains. Some genomic loci have been proven difficult to knock out. Reasons might be the presence of repetitive sequences, extensive DNA methylation, or heterochromatin. The confounding presence of neighboring 129 genes on the knockout section of genetic material has been christened with the name “flanking-gene effect”. Methods and guidelines to deal with this issue have been proposed.
Another limitation is that conventional knockout mice develop in the absence of the gene being investigated. At times, loss of activity during development might mask the role of the gene in the adult state, particularly if the gene is involved in a number of processes spanning development. Conditional mutation approaches are then needed that first enable the mouse to develop and mature normally before ablation of the gene of interest.
Another serious limitation is a lack of evolution adaptations in the knockout model that may take place in wild type animals after they naturally mutate.
Image Caption: A laboratory mouse in which a gene affecting hair growth has been knocked out (left), is shown next to a normal lab mouse. Credit: Maggie Bartlett, NHGRI/Wikipedia