Knockout Mouse - Procedure

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 neighbour sequence, except that it is changed sufficiently to make the gene inoperable. Usually, the new sequence is also given a marker gene, a gene that normal mice don't have and that confers resistance to a certain toxic agent or that produces an observable change (e.g. colour or fluorescence).
2. Stem cells are isolated from a mouse blastocyst (a very young embryo) and grown in vitro. For this example, we will take stem cells from a white mouse.
3. The new sequence from step 1 is introduced into the stem cells from step 2 by electroporation. The natural process of homologous recombination means some of the electroporated stem cells will incorporate the new sequence with the knocked-out gene into their chromosomes in place of the original gene. The chances of a successful recombination event are relatively low, so the majority of altered cells will have the new sequence in only one of the two relevant chromosomes - they are said to be heterozygous.
4. The stem cells that incorporated the knocked-out gene are isolated from the unaltered cells using the marker gene from step 1. For example, the unaltered cells can be killed using a toxic agent to which the altered cells are resistant.
5. The knocked-out stem cells from step 4 are inserted into a mouse blastocyst. For this example, we use blastocysts from a grey mouse. The blastocysts now contain two types of stem cells: the original ones (from the grey mouse), and the knocked-out cells (from the white mouse). These blastocysts are then implanted into the uterus of female mice, where they develop. The newborn mice will therefore be chimeras: some parts of their bodies result from the original stem cells, other parts from the knocked-out stem cells. Their fur will show patches of white and grey, with white patches derived from the knocked-out stem cells and grey patches from the recipient blastocyst.
6. Some of the newborn chimera mice will have gonads derived from knocked-out stem cells, and will therefore produce eggs or sperm containing the knocked-out gene. When these chimera mice are crossbred with others of the wild type, some of their offspring will have one copy of the knocked-out gene in all their cells. These mice will be entirely white and are not chimeras, however they are still heterozygous.
7. When these heterozygous offspring are interbred, some of their offspring will inherit the knocked-out gene from both parents; they carry no functional copy of the original gene (i.e. they are homozygous for that allele).

A detailed explanation of how knockout (KO) mice are created is located at the website of the Nobel Prize in Physiology or Medicine 2007.