Knockout mouse

A knockout mouse is a genetically engineered mouse one or more of whose genes have been made inoperable. Knockout is a route to learning about a gene that has been sequenced but has an unknown or incompletely known function. (The general process of knocking out genes is explained in the article on gene knockout.)

Mice are the laboratory animal species most closely related to humans in which the knockout technique can be easily performed, so they are a favorite subject for knockout experiments, especially with regard to genetic questions that relate to human physiology. (Gene knockout in rats is much harder and has only been possible since 2003.) Knockout mice are frequently used in drug development: a gene is disabled to model a certain human disease in mice; then the effectiveness of different drug candidates can be tested on those mice. Or, a candidate drug target can be evaluated by knockout. This simulates the effect of a drug before the the drug itself has been discovered. This works for drugs that act by chemically inhibiting a target.

Knockout mice can be patented in many countries, including the United States.

The first knockout mice were produced by Mario Capecchi, Martin Evans and Oliver Smithies in 1987-1989.

Procedure

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

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 neighbor sequence, except that it is changed sufficiently to make it inoperable. Usually, the new sequence is also given a marker gene, a gene that normal mice don't have and that transfers resistance to a certain antibiotic.

2. From a mouse blastocyte (a very young embryo consisting of a ball of undifferentiated cells), stem cells are isolated; these can be grown in vitro. For this example, we will take a blastocyte from a white mouse.

3. The stem cells from step 2 are combined with the new sequence from step 1. This is done via electroporation (using electricity to transfer the DNA across the cell membrane). Some of the electroporated stem cells will incorporate the new sequence into their chromosomes in place of the old gene; this is called homologous recombination. The reason for this process is that the new and the old sequence are very similar. Using the antibiotic from step 2, those stem cells that actually did incorporate the new sequence can be quickly isolated from those that did not.

4. The stem cells from step 3 are inserted into mouse blastocytes. For this example, we use blastocytes from a grey mouse. These blastocytes are then implanted into the uterus of female mice, to complete the pregnancy. The blastocytes contain two types of stem cells: the original ones (grey mouse), and the newly engineered ones (white mouse). The newborn mice will therefore be chimeras: parts of their bodies result from the original stem cells, other parts result from the engineered stem cells. Their furs will show patches of white and grey.

5. Newborn mice are only useful if the newly engineered sequence was incorporated into the germ cells (egg or sperm cells). So we cross these new mice with others and watch for offspring that's all white. These are then further inbred to produce mice that carry no functional copy of the original gene.

Procedure

See also