Knockout mice - germline transmission

Knockout mice

8/1/03. By Richard Twyman

The inactivation of a specific gene in a mouse.

Knockout mice contain the same, artificially introduced mutation in every cell, abolishing the activity of a preselected gene. The resulting mutant phenotype (appearance, biochemical characteristics, behaviour etc.) may provide some indication of the gene's normal role in the mouse, and by extrapolation, in human beings. Knockout mouse models are widely used to study human diseases caused by the loss of gene function.

Key principles

  • A mutant version of the preselected target gene is constructed in the laboratory.
  • Knockout is achieved by swapping the functional copy of the gene for the mutated version in mouse embryonic stem cells (ES cells).
  • Mice are derived from the modified ES cells. These mice carry the same mutation in every cell. An additional round of breeding is required to produce mice that are homozygous for the mutation.
  • In traditional constitutive knockout mice the mutation is present throughout development and in all cells of the adult. Conversely, in conditional knockout mice, other genetic strategies are incorporated that allow mutations to be induced at different stages of development or in selected cell types.

How does it work?

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Knockout mice contain an artificially introduced mutation in their cells, abolishing the activity of a preselected gene.
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The gene targeting process is carried out in mouse embryonic stem cells (ES cells). These cells are derived from a very early (usually male) mouse embryo and can therefore differentiate into all types of cell when introduced into another embryo.
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A mutant version of the target gene is introduced into the ES cells.
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Recombination between the related sequences of DNA swaps the mutated version of the target gene into the mouse genome.
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The ES cells need a host embryo in which to develop, and this is isolated from a mouse with different coloured fur. The use of mice with different coat colours for the ES cells and the host embryo allows the mutant gene to be followed.
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The ES cells with one mutant gene are transferred into the host embryo.
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The resulting mouse is a chimera, as has developed from a mixture of cells from brown and black mice. If the ES cells with the mutant gene contribute to the germline, the next generation of mice have one nonfunctional copy of the gene.
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Inbreeding produces knockout mice where both copies of the gene are mutated.

Knockout mice are produced by a technique called gene targeting. This is the replacement of one gene sequence, the sequence resident in the mouse genome, with a related sequence that has been modified in the laboratory to contain a mutation.

The replacement occurs by a process called homologous recombination, where two very similar DNA sequences line up next to each other and exchange parts.

Gene targeting is carried out in mouse embryonic stem cells (ES cells). These cells are derived from a very early (usually male) mouse embryo and can therefore differentiate into all types of cell when introduced into another embryo. The aim is to get the modified ES cells to contribute to the germ line, which gives rise to sperm. Some sperm are produced that carry the desired mutation, and if these fertilise a normal egg, mice develop with one copy of the mutated gene in every cell.

Interbreeding such mice will produce some homozygous individuals in the next generation - mice inheriting the mutation from both parents and therefore carrying two copies of the mutant gene. These are knockout mice.

How is it used?

The phenotype of a knockout mouse provides important clues about the gene's normal role. One major application of this technology is the modelling of human diseases caused by loss of gene function. Examples include cystic fibrosis, beta-thalassaemia and various forms of cancer. Such models are useful because they can be used to investigate the biochemical and physiological aspects of the disease and for the development and testing of drugs.

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