Gene Knockout (KO)

A gene knockout is a genetic technique in which one of an organism's genes is made

inoperative. Also known as knockout organisms or simply knockouts, they are used in
learning about a gene that has been sequenced, but which has an unknown or incompletely
known function. Researchers draw inferences from the difference between the knockout
organism and normal individuals.

Use
Knocking out the activity of a gene provides information about what that gene normally does.
Humans share many genes with mice. Consequently, observing the characteristics of
knockout mice gives researchers information that can be used to better understand how a
similar gene may cause or contribute to disease in humans.
Examples of research in which knockout mice have been useful include studying and
modeling different kinds of cancer, obesity, heart disease, diabetes, arthritis, substance
abuse, anxiety, aging and Parkinson's disease. Knockout mice also offer a biological and
scientific context in which drugs and other therapies can be developed and tested.
Millions of knockout mice are used in experiments each year.

Method
Knockout is accomplished through a combination of techniques, beginning in the test tube
with a plasmid, a bacterial artificial chromosome or other DNA construct, and proceeding to
cell culture. Individual cells are genetically transformed with the DNA construct. Often the goal
is to create a transgenic animal that has the altered gene. If so, embryonic stem cells are
genetically transformed and inserted into early embryos. Resulting animals with the genetic
change in their germline cells can then often pass the gene knockout to future generations.

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 it 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). The chances of a successful
recombination event are relatively low, so the majority of altered cells will have the
gene changed for only one of the two relevant chromosomes - they are said to be
heterozygous.
2. From a mouse blastocyst (a very young embryo consisting of a ball of undifferentiated
cells with surrounding extra-embryonic cells), stem cells are isolated; these can be
grown in vitro. For this example, we will take a stem cell 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 sequences are
very similar. Using the marker gene from step 1, 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 a mouse blastocyst. For this example, we
use blastocysts from a grey mouse. These blastocysts are then implanted into the
uterus of female mice, to complete the pregnancy. The blastocysts 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). These new mice are crossed with others of the
white type for offspring that are all white. These mice still contain one functional copy of
the gene and must be further inbred to produce mice that carry no functional copy of
the original gene (i.e. are homozygous for that allele).
Breeding scheme for producing knockout mice. Blastocysts containing cells, that are both
wildtype and knockout cells, are injected into the uterus of a foster mother. This produces
offspring that are either wildtype and coloured the same colour as the blastocyst donor (grey)
or chimera (mixed) and partially knocked out. The chimera mice are crossed with a normal
wildtype mouse (grey). This produces offspring that are either white and heterozygous for the
knocked out gene or grey and wildtype. White heterozygous mice can subsequently be
crossed to produce mice that are homozygous for the knocked out gene.