Knockout mouse
A knockout mouse ( Engl. Knock out "incapacitate") or KO mouse is a mouse ( Mus musculus ), in which genetic means of a manipulation ( gene targeting ) targeted one or more genes have been turned off ( gene knockout ) . This manipulation takes place on the embryonic stem cells (from mice), which are then introduced into the germline of a mouse. There are now knockout mice for a wide variety of research areas. With the help of the genetically modified animals, biological mechanisms can be examined, for example. They are also suitable as models for human diseases and for pharmacological questions.
For their work on knockout mice, the 2007 Nobel Prize in Physiology or Medicine was awarded to Martin Evans , Mario Capecchi and Oliver Smithies .
method
Embryonic stem cells are removed from blastocysts of an inbred mouse strain and propagated in vitro . An inactivation vector is now transferred into the still undifferentiated stem cells by electroporation , microinjection or another suitable method. The inactivation vector is produced artificially and consists of the gene to be inactivated, which carries a mutation so that it can no longer be transcribed, or the resulting protein is inactive. The exchange between the DNA segments takes place through homologous recombination . In homologous recombination, the neighboring sections of the gene on the vector are stored at the same location in the mouse genome and are in some cases recombined .
A positive marker is usually added to the sequence . Usually a neomycin resistance gene is chosen here. This allows you to later see by administering neomycin (an antibiotic ) which cells have incorporated the sequence and which have not. Only cells with the neomycin resistance survive.
The gene for the thymidine kinase (HSV-tk) of the herpes simplex virus (HSV) is used as a marker to test whether the incorporation has taken place in the right place in the mouse genome . During subsequent treatment with ganciclovir (a virostat), if the HSV-tk cassette is integrated, a product is formed which inhibits the replication of the cells. Integration occurs as a result of the non-targeted recombination, since the selection cassette flanks the homology construct but not in the case of homologous recombination. After homologous recombination, the cells survive treatment with ganciclovir. This ensures that the installation took place at a defined point.
The recombined stem cells are inserted into a blastocyst , which in turn is implanted into a pretreated recipient mouse. Mixed-cell animals ( chimeras ) then develop in the surrogate mother . The heterozygous animals can be filtered out by backcrossing against the wild type . Through intersections obtained homozygous animals, so where destroyed the desired gene in all cells - knocked out is.
The Cre / loxP system is used in today's common application . In a first step, loxP - DNA sequences are introduced by means of homologous recombination. As a rule, in order not to disturb gene expression , these are inserted into introns which flank the exons to be deleted. These modified animals are bred in the next step with a mouse as a transgene , the Cre recombinase expressed. In this way, it is possible to generate deletions specific to tissue and development. Another advantage is the possibility of deleting the neomycin cassette in the embryonic stem cells. The neomycin cassete often interferes with the expression of neighboring genes.
The method itself has changed so that the manipulation can be carried out directly in the fertilized egg cell without prior selection of recombinant cells. This was made possible by the development of molecular gene scissors such as transcription activator-like effector nucleases and zinc finger nucleases , but in particular the CRISPR / Cas method . The CRISPR / Cas system made it possible to shorten the production time of knockout mice considerably.
Examples
BSE resistant mice
To confirm the theory of the future Nobel Prize winner Stanley Prusiner , according to which diseases such as bovine spongiform encephalopathy (BSE), Creutzfeldt-Jakob disease and the like. a. caused by endogenous proteins, the so-called prions or their changed forms, a knockout mouse with a defective prion gene was created by Charles Weissmann in Zurich ( PrP - / PrP - ). Although the prion protein is present in the brain of normal mice, the knockout mice appear to be fully viable without the Prp gene product. However, these knockout mice are resistant to infection with otherwise deadly prions, so that there is the possibility of z. B. also to breed cattle with the resistance to mad cow disease.
Biological mechanisms
In chronobiology , knockout mice are used to understand the molecular mechanisms behind the circadian rhythm . By specifically switching off certain genes and thus their expression , it is possible to determine which place these genes and the proteins encoded by them occupy in the circadian rhythm on the basis of changes in the behavior of the mice in the rhythm of the day.
medicine
In many human diseases , the background is an impaired gene function. The knockout mice are an ideal disease model here. The ability to quickly breed knockout mice makes it possible to have an animal model available to make statements about the role of certain genes in diseases and their treatments. However, the transferability of the results is always problematic.
criticism
In addition to the indisputable benefit for research, the knock-out process is also the subject of criticism, as it is, by definition, tied to animal experiments . For a more detailed discussion of the ethical criticisms see there.
See also
literature
- Jochen Graw (2007): Nobel Prize 2007 in Medicine: Making knockout mice. In: Biology in Our Time. 37 (6): 352-354. PDF
- Braun, R. & Willnow, E. (1996): The Knockout Mouse as a Disease Model: Principles and Clinical Relevance. In: Deutsches Ärzteblatt. Vol. 93, No. 26, p. 1765.
Web links
- Live longer thanks to the knockout mouse. Telepolis from January 26, 2003.
- Nobel Prizes 2007 - Cooperative gene failure idea. Spectrumdirect from October 8, 2007.
- Homologous recombination method, and knockout mice (English)
Individual evidence
- ↑ Information from the Nobel Foundation on the 2007 award ceremony for Martin Evans, Mario Capecchi and Oliver Smithies (English)
- ↑ K. Rana, MV Clarke, JD Zajac, RA Davey, HE MacLean: Normal phenotype in conditional androgen receptor (AR) exon 3-floxed neomycin-negative male mice. In: Endocr. Res. Volume 39, number 3, 2014, pp. 130-135, doi : 10.3109 / 07435800.2013.864303 , PMID 24467187 .
- ↑ H. Wang, H. Yang, CS Shivalila, MM Dawlaty, AW Cheng, F. Zhang, R. Jaenisch: One-step generation of mice carrying mutations in multiple genes by CRISPR / Cas-mediated genome engineering. In: Cell. Volume 153, Number 4, May 2013, pp. 910-918, ISSN 1097-4172 . doi: 10.1016 / j.cell.2013.04.025 . PMID 23643243 . PMC 3969854 (free full text).