What is a gene knockout

Importance of knock-out mice

Production and importance of knock-out mice

If one wants to examine the function of a gene, one looks for mutants in this gene. These are then compared with normal animals that do not have such a mutation. However, it can never be completely ruled out that the differences are at least partly due to other genes, in which the animals inevitably differ. The principle of isolating variation could therefore not be strictly adhered to in biology.
Knock-out mice are those animals in which a very specific gene has been specifically switched off in order to test its effects. One would like to have two strains of mice that are the same in all genes and only differ in the knock-out gene. In the first trunk it is intact, in the second it is defective. Then differences in body structure or physiology must be attributed to this gene. Therefore, a strain of mice in which all animals have the same genetic background is required for this experiment. Inbred animals are therefore used. This inbreeding has been carried out for several hundred generations (consistent brother-sister mating), which means that the animals do not differ genetically at all or only very slightly.
In addition, mutants of a gene are often difficult to find and identify. While it is easy with organisms such as Drosophila to generate and study many different mutants e.g. by irradiation, this is not possible with mammals because of their low number of offspring. With this group of animals, you are largely dependent on chance to discover a certain mutation. For example, if you look for mice that have a disease similar to cystic fibrosis, a mutation in the gene occurs very rarely and may also be difficult to find. The fact that mice can specifically mutate in certain genes can be used to create so-called disease models for certain diseases.

The steps that are necessary for the production of transgenic knock-out mice should now be put together here and each step should be explained in more detail.
1. Stem cells from mice are used to generate the mutates, because naturally not all body cells can be changed in an adult mouse. Such stem cells are taken from blastocysts (see stem cells). The removed stem cells can be multiplied and kept alive in vitro by adding growth factors.

a: The gene that is to be changed or mutated. E1 to E5 are exons, with introns in between.
b: The red arrows show which parts of the introduced DNA are homologous to the original gene. neo is the neomycin resistance gene, HSVTk is a section that codes for a thymidine kinase and is also integrated if no homologous recombination takes place. LxP are sequences, which are not explained in detail here, which are used to cut out the neomycin resistance gene (they are not absolutely necessary for the method).
c: This is what the DNA sequence looks like in the animal after it has been changed. The gene can no longer be read because the neomycin sequence lies between the exons. (It must be ensured that the gene is really not read, e.g. by inserting a polyA sequence in front of the neomycin gene. The neomycin gene needs its own strong promoter to ensure that it is also read.).
d: The neomycin gene can be cut out with the Cre recombinase. The remaining gene is defective because it lacks exon 2.

2. These stem cells are now genetically modified by so-called homologous recombination. This is a process by which any other sequence can be used at a specifically selected location in the genome. For example, you can switch off the insulin gene by inserting a completely different sequence of nucleotides into this gene, which means that functional insulin can no longer be formed. The sequence is used by producing it artificially and then introducing it into the stem cell via electroporation (the membrane is made so permeable through an electric current that the artificial DNA molecules can penetrate). The cell then integrates into the genome itself. However, the sequence can on the one hand be used in the right place, but on the other hand it can also be used in an undesired place or even several times, which is not what you want. To rule this out, the sequence used is given three important properties (see figure). It contains a marker (usually the neomycin resistance gene) which is used to later identify the cells as positive (when neomycin is added, all cells that have not integrated the sequence die) that have the altered genetic material. Around the marker on each side there are sequences that are homologous to the intact gene, which are intended to ensure that hybridization with the genetic material takes place here and that the sequence can thus be used at the right place in the genetic material. At the end a second marker is used for a negative selection. In the case of homologous recombination, this is not integrated into the genome, but it is if the sequence is integrated into the genome at any other point. This marker is therefore used to distinguish the cells in which a homologous recombination has occurred from those in which a random integration has occurred (or multiple integrations have taken place). The cells where the sequence has integrated in the wrong place are now killed. This leaves only stem cells that have mutated at the desired location in the genome.

3. The genetically modified stem cell is now inserted into another blastocyst, which in turn is implanted into a pseudopregnant mouse, which then carries the growing animal to term. From this implanted blastocyst a chimeric animal emerges, as they say, an animal whose body cells do not all contain the same genetic material, but which on the one hand contains cells with the original genetic material and on the other hand those with the genetic material of the stem cell used (the body is therefore like a mosaic of two different genetic types).
It can now be determined that the stem cell used contributes to all tissues of the growing mouse. This means that the mouse also has egg or sperm cells with the changed genetic material.

4. The chimeric animal is now crossed with a normal animal. The offspring are mostly homozygous (healthy), but some of them carry the modified gene because the egg or sperm cell is derived from the stem cell used. You are heterozygous. These heterozygous animals now have to be found out, which is done by examining the genetic make-up. From such heterozygous animals one now needs a female and a male. These are crossed with each other. 25% of the offspring of this couple must be homozygous. The set goal has thus been achieved.

Great advances have been made in studying development by this method. E.g. the genes Myf5 and MyoD are necessary for the development of muscles. It then turned out that both genes need to be knocked out so that no muscles develop. This leads to the conclusion that these genes can partially replace each other, i.e. have redundant effects.
For the study of cystic fibrosis, a "mouse model" was produced which carries the same mutation as 70% of the patients, which has led to great advances in treatment.
Mice whose p53 gene is mutated develop cancer. They are a very useful model for cancer research. It then turned out that 50% of all cancer patients have a mutation in this gene, which shows the enormous importance of this gene for the development of cancer.

I would like to thank Mr. Euwens (Uni Bonn) for pointing out the importance of using inbred strains and that it is not a matter of course that the gene is not read after homologous recombination.

Address: Helmut Hupfeld, Katzenberg 11, 27283 Verden, [email protected]