Knockout mouse

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A 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 replacing it or disrupting it with an artificial piece of DNA. Knockout mouse_sentence_0

They are important animal models for studying the role of genes which have been sequenced but whose functions have not been determined. Knockout mouse_sentence_1

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 function. Knockout mouse_sentence_2

Mice are currently the laboratory animal species most closely related to humans for which the knockout technique can easily be applied. Knockout mouse_sentence_3

They are widely used in knockout experiments, especially those investigating genetic questions that relate to human physiology. Knockout mouse_sentence_4

Gene knockout in rats is much harder and has only been possible since 2003. Knockout mouse_sentence_5

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. Knockout mouse_sentence_6

Aspects of the technology for generating knockout mice, and the mice themselves have been patented in many countries by private companies. Knockout mouse_sentence_7

Use Knockout mouse_section_0

Knocking out the activity of a gene provides information about what that gene normally does. Knockout mouse_sentence_8

Humans share many genes with mice. Knockout mouse_sentence_9

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. Knockout mouse_sentence_10

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 mouse_sentence_11

Knockout mice also offer a biological and scientific context in which drugs and other therapies can be developed and tested. Knockout mouse_sentence_12

Millions of knockout mice are used in experiments each year. Knockout mouse_sentence_13

Strains Knockout mouse_section_1

There are several thousand different strains of knockout mice. Knockout mouse_sentence_14

Many mouse models are named after the gene that has been inactivated. Knockout mouse_sentence_15

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 and/or inducing apoptosis. Knockout mouse_sentence_16

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. Knockout mouse_sentence_17

Other mouse models are named according to their physical characteristics or behaviours. Knockout mouse_sentence_18

Procedure Knockout mouse_section_2

There are several variations to the procedure of producing knockout mice; the following is a typical example. Knockout mouse_sentence_19

Knockout mouse_ordered_list_0

  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.Knockout mouse_item_0_0
  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.Knockout mouse_item_0_1
  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.Knockout mouse_item_0_2
  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.Knockout mouse_item_0_3
  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.Knockout mouse_item_0_4
  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.Knockout mouse_item_0_5
  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).Knockout mouse_item_0_6

A detailed explanation of how knockout (KO) mice are created is located at the website of the Nobel Prize in Physiology or Medicine 2007. Knockout mouse_sentence_20

Limitations Knockout mouse_section_3

The National Institutes of Health discusses some important limitations of this technique. Knockout mouse_sentence_21

There is variability in the whole procedure depending largely on the strain from which the stem cells have been derived. Knockout mouse_sentence_22

Generally cells derived from strain 129 are used. Knockout mouse_sentence_23

This specific strain is not suitable for many experiments (e.g., behavioural), so it is very common to backcross the offspring to other strains. Knockout mouse_sentence_24

Some genomic loci have been proven very difficult to knock out. Knockout mouse_sentence_25

Reasons might be the presence of repetitive sequences, extensive DNA methylation, or heterochromatin. Knockout mouse_sentence_26

The confounding presence of neighbouring 129 genes on the knockout segment of genetic material has been dubbed the "flanking-gene effect". Knockout mouse_sentence_27

Methods and guidelines to deal with this problem have been proposed. Knockout mouse_sentence_28

Another limitation is that conventional (i.e. non-conditional) knockout mice develop in the absence of the gene being investigated. Knockout mouse_sentence_29

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. Knockout mouse_sentence_30

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. Knockout mouse_sentence_31

Another serious limitation is a lack of evolutive adaptations in knockout model that might occur in wild type animals after they naturally mutate. Knockout mouse_sentence_32

For instance, erythrocyte-specific coexpression of GLUT1 with stomatin constitutes a compensatory mechanism in mammals that are unable to synthesize vitamin C. Knockout mouse_sentence_33

See also Knockout mouse_section_4

Knockout mouse_unordered_list_1

Credits to the contents of this page go to the authors of the corresponding Wikipedia page: mouse.