Knockout mouse

A knockout mouse (English knock -out - incapacitate ) is a mouse which, by means of genetic manipulation ( gene targeting, gene targeting English ) targeted one or more genes have been disabled ( gene knockout ). This manipulation is done on embryonic stem cells ( mice ), which are then introduced into the germline of a mouse. There are now knockout mice for a variety of research areas. With the aid of genetically modified animals biological mechanisms may for example be examined. They are also suitable as models for human diseases or pharmacological issues.

For her work on knockout mice of the Nobel Prize in Physiology or Medicine was awarded to Martin Evans, Mario Capecchi and Oliver Smithies, 2007.

Method

From blastocysts one inbred mouse strain embryonic stem cells are taken and propagated in vitro. Now an inactivation is transferred by electroporation, microinjection or any other suitable method in the undifferentiated stem cells. The inactivation is synthesized and is made to be inactivated to the gene carries a mutation such that it can not be transcribed, or the resulting protein is inactive. The exchange between the DNA sections via homologous recombination. In homologous recombination, the adjacent portions of the gene on the vector stored at the same location in the mouse genome and are recombined in some cases.

In general, the sequence of a positive marker is still attached. Usually one chooses a neomycin resistance gene here. Characterized it has the possibility to see later by administration of neomycin (an antibiotic), which cells have incorporated the sequence and which are not. Only cells with the neomycin resistance survive.

In order to test whether the replacement is made ​​at the correct place in the mouse genome, the gene for thymidine kinase ( HSV-tk ) of Herpes simplex virus (HSV ) is used as a marker. Upon subsequent treatment with ganciclovir ( a virostatic ) a product is formed, which inhibits the replication of the cells. However, when the HSV-tk frame is destroyed by the incorporation of the desired insert, the cells survive the treatment with ganciclovir. This ensures that the fitting is passed at a defined location.

The recombinant stem cells can be used into a blastocyst that is implanted in turn a pretreated recipient mouse. In the surrogate then mixed- animals develop ( chimeras ). By backcrossing to the wild type, the heterozygous animals can be filtered out. Intersections obtained homozygous animals, in which therefore destroys the desired gene in all cells - has been knocked out.

Examples

BSE -resistant mice

To confirm the theory of the later Nobel laureate Stanley Prusiner, after diseases such as mad cow disease (BSE ), Creutzfeldt -Jakob disease, among others by endogenous proteins, known as prions, or their modified forms, caused a KO mouse was by Charles Weissmann in Zurich with a defective prion gene ( PrP-/PrP- ). Although the prion protein in the brain of normal mice is present, the KO mice appear to be fully viable without the PrP gene. These KO mice, however, are against the otherwise lethal infection with prions resistant, so this results in the possibility of such also cattle with resistance breeding against mad cow disease.

Biological Mechanisms

In chronobiology knockout mice are used to understand the molecular mechanisms that underlie the circadian rhythm. By specifically knocking out certain genes and thus their expression can be based mooring of circadian changes in behavior of the mice, which are taking place these genes and the proteins encoded by them in the circadian rhythm.

Medicine

In many human diseases, the background is a disturbed gene function. The knockout mice are an ideal disease model here. By being able to breed knockout mice quickly, it is possible to have an animal model available to make statements about the role of certain genes in diseases and their treatments. The problem, however, is always the transferability of the results.

Criticism

In addition to the undeniable benefit to the research, the knock-out process is also subject to criticism because it is bound by definition to animal testing. A more detailed discussion of the ethical criticisms see there.