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Gene knockout
A gene knockout is a genetically engineered organism that carries one or more genes in its
chromosomes that have been made inoperative (have been "knocked out" of the organism). This is
done for research purposes. 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.
The term also refers to the process of creating such an organism, as in "knocking out" a gene.
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 construct and--for knockouts in multi-cellular
organisms--ultimately fused with a stem cell from a nascent embryo.
The construct is engineered to recombine with the target gene, which is accomplished by
incorporating sequences from the gene itself into the construct. Recombination then occurs in the
region of that sequence within the gene, resulting in the insertion of a foreign sequence to disrupt
the gene. With its sequence interrupted, the altered gene in most cases will be translated into a nonfunctional protein, if it is translated at all.
A conditional knockout allows gene deletion in a tissue specific manner.
Because recombination is a rare event in the case of most cells and most constructs, the foreign
sequence chosen for insertion usually is a reporter. This enables easy selection of cells or
individuals in which knockout was successful.
In diploid organisms, which contain two alleles for most genes, and may as well contain several
related genes that collaborate in the same role, additional rounds of transformation and selection are
performed until every targeted gene is knocked out.
Knock-in is similar to knock-out, but instead it replaces a gene with another instead of deleting it.
Knockout mouse
A knockout mouse is a genetically engineered mouse one or more of whose genes have been made
inoperable through a gene knockout. Knockout is a route to learning about a gene that has been
sequenced but has an unknown or incompletely known function. Mice are the laboratory animal
species most closely related to humans in which the knockout technique can be easily performed, so
they are a favourite 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.)
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
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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 modelling
different kinds of cancer, obesity, heart disease, diabetes, arthritis, substance abuse, anxiety, aging
and Parkinson disease. Knockout mice also offer a biological context in which drugs and other
therapies can be developed and tested.
Many of these mouse models are named after the gene that has been inactivated. For example, the
p53 knockout mouse is named after the p53 gene which codes for a protein that normally suppresses
the growth of tumours by arresting cell division. Humans born with mutations that inactivate the
p53 gene suffer from Li-Fraumeni syndrome, a condition that dramatically increases the risk of
developing bone cancers, breast cancer and blood cancers at an early age. Other mouse models are
named, often with creative flair, according to their physical characteristics or behaviours. For
example, "Methuselah" is a knockout mouse model noted for longevity, while "Frantic" is a model
useful for studying anxiety disorders.
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 or a selectable marker.
2. From a mouse morula (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 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 sequence are very similar. Using the antibiotic 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 mouse blastocyst cells. 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). So we cross these new mice with others and watch for
offspring that are all white. These are then further inbred to produce mice that carry no
functional copy of the original gene.
Limitations
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While knockout mice technology represents a valuable research tool, some important limitations
exist. 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.
There is variability in the whole procedure depending largely on the strain from which the stem
cells have been derived. Generally cells derived from strain 129 are used. This specific strain is not
suitable for many experiments (e.g., behavioural), so it is very common to backcross the offspring
to other strains. Some genomic loci have been proven very difficult to knock out. Reasons might be
the presence of repetitive sequences, extensive DNA methylation, or heterochromatin.
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