A CRISPR-Cas9 mediated knockout mouse or knock-out mouse is a genetically modified mouse (Mus musculus) in which researchers have inactivated, or "knocked out", an existing gene by disrupting it or replacing it with an artificial piece of DNA. They are important animal models for studying the functions of genes. By causing a specific gene to be inactive in the mouse, and observing any differences from normal behaviour or physiology, researchers can infer its probable functions.
Knocking out the activity of a gene provides information about what that gene normally does. Humans share many genes with mice that are currently the laboratory animal species most closely related to the humans. The knockout technique can easily be applied to 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.
The first recorded knockout mouse was created by Mario R. Capecchi, Martin Evans, and Oliver Smithies in 1989, for which they were awarded the 2007 Nobel Prize in Physiology or Medicine. Now millions of knockout mice are used in experiments each year . Gene knockout in rats is much harder and has only been possible since 2003 .
There are several different procedures 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 the gene 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 (e.g., neomycin) or that produces an observable change (e.g. colour or fluorescence). In addition, a second gene, such as herpes tk+, is also included in the construct in order to accomplish a complete selection.
2. Embryonic stem cells are isolated from a mouse blastocyst(a very young embryo) and grown in vitro. For this example, we will take stem cells from a white mouse.
3. The new sequence from step 1 is introduced into the stem cells from step 2 by 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 relatively 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.
Cells that were transformed with a vector containing the neomycin resistance gene and the herpes tk+ gene are grown in a solution containing neomycin and Ganciclovir in order to select for the transformations that occurred via homologous recombination. Any insertion of DNA that occurred via random insertion will die because they test positive for both the neomycin resistance gene and the herpes tk+ gene, whose gene product reacts with Ganciclovir to produce a deadly toxin. Moreover, cells that do not integrate any of the genetic material test negative for both genes and therefore die as a result of poisoning with neomycin.
Fig 1. Procedure of CRISPR-Cas9 Knockout Mouse.
4. The embryonic stem cells that incorporated the knocked-out gene are isolated from the unaltered cells using the marker gene from step 1. For example, the unaltered cells can be killed using a toxic agent to which the altered cells are resistant.
5. The knocked-out embryonic stem cells from step 4 are inserted into a mouse blastocyst. For this example, we use blastocysts from a grey mouse. The blastocysts now contain two types of stem cells: the original ones (from the grey mouse), and the knocked-out cells (from the white mouse). These blastocysts are then implanted into the uterus of female mice, 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 stem cells. Their fur will show patches of white and grey, with white patches derived from the knocked-out stem cells and grey patches from the recipient blastocyst.
6. 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. These mice will be entirely white and are not chimeras, however they are still heterozygous.
7. 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 (i.e. they 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 .
1. Spencer G (December 2002). Background on Mouse as a Model Organism. National Human Genome Research Institute.
2. Pilcher HR (2003.05.19). It's a knockout. Nature. doi:10.1038/news030512-17.
3. Zan Y et al. (June 2003). Production of knockout rats using ENU mutagenesis and a yeast-based screening assay. Nature Biotechnology. 21(6): 645–51. doi:10.1038/nbt830.
4. The Nobel Prize in Physiology or Medicine 2007. Nobelprize.org. 1985.09.19.