GENE KNOCKOUT

A gene knockout is a genetic technique in which an organism is engineered to carry genes that have
been made inoperative (have been "knocked out" of the organism)

Knockout mouse

A knockout mouse is a genetically engineered mouse in which one or more genes have been
turned off through a targeted mutation.

 Knockout mice are important animal models for studying the role of genes which have
been sequenced but whose functions have not been determined. By causing a specific
gene to be inactive in the mouse, and observing any differences from normal behaviour
or condition, researchers can infer its probable function.

 Mice are currently the most closely related laboratory animal species to humans for
which the knockout technique can easily be applied. They are widely used in knockout
experiments, especially those investigating genetic questions that relate to human
physiology.

 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.

A knockout mouse (left) that is a model of A laboratory mouse in which a gene affecting hair growth has
obesity, compared with a normal mouse. been knocked out (left), is shown next to a normal lab mouse

Procedure of producing Knock out Mouse

 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)

Limitations

About 15 percent of gene knockouts are developmentally lethal, which means that the genetically
altered embryos cannot grow into adult mice. The lack of adult mice limits studies to embryonic
development and often makes it more difficult to determine a gene's function in relation to
human health. In some instances, the gene may serve a different function in adults than in
developing embryos.

Knocking out a gene also may fail to produce an observable change in a mouse or may even
produce different characteristics from those observed in humans in which the same gene is
inactivated. For example, mutations in the p53 gene are associated with more than half of human
cancers and often lead to tumours in a particular set of tissues. However, when the p53 gene is
knocked out in mice, the animals develop tumours in a different array of tissues.
GENE TARGETING

Gene targeting (a replacement strategy based on homologous recombination) is a genetic

technique that uses homologous recombination to change an endogenous gene. The method can
be used to delete a gene, remove exons, add a gene, and introduce point mutations. Gene
targeting can be permanent or conditional. Conditions can be a specific time during development
/ life of the organism or limitation to a specific tissue. Gene targeting requires the creation of a
specific vector for each gene of interest. However, it can be used for any gene, regardless of
transcriptional activity or gene size.

Methods

 A targeting construct made out of DNA is generated in bacteria. It typically contains part
of the gene to be targeted, a reporter gene, and a (dominant) selectable marker.
 To target genes in mice, this construct is then inserted into mouse embryonic stem cells in
culture. After cells with the correct insertion have been selected, they can be used to
contribute to a mouse's tissue via embryo injection. Finally, chimeric mice where the
modified cells made up the reproductive organs are selected for via breeding. After this
step the entire body of the mouse is based on the previously selected embryonic stem cell.
 To target genes in moss, this construct is incubated together with freshly isolated
protoplasts and with Polyethylene glycol. As mosses are haploid organisms [2],
regenerating moss filaments (protonema) can directly be screened for gene targeting,
either by treatment with antibiotics or with PCR. Unique among plants, this procedure for
reverse genetics is as efficient as in yeast [3]. Using modified procedures, gene targeting
has also been successfully applied to cattle, sheep, swine, and many fungi.

Applications

Gene targeting has been widely used to study human genetic diseases by removing "knock-out",
or adding "knock-in", specific mutations of interest to a variety of models. Previously used to
engineer rat cell models, advances in gene targeting technologies are enabling the creation of a
new wave of isogenic human disease models. These models are the most accurate in-vitro
models available to researchers to date, and are facilitating the development of new personalised
drugs and diagnostics, particularly in the field of cancer.