PRODUCTION OF KNOCK- OUT MICE

SUBMITTED TO: SUBMITTED BY:

DR. GIRISH SHARMA TANYA MALIK

M.Sc. Biotechnology (2ndsem)

MSB/21/151
 KNOCK OUT
A knockout mouse, or knock-out mouse, is a genetically modified mouse in which researchers
have inactivated, or "knocked out: an existing gene by replacing it or disrupting it with an
artificial piece of DNA. They 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 behavior or
physiology, researchers can infer its probable function.

Mice are currently the laboratory animal species most closely related 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. gene knock
out in rats is much harder and has only been possible since 2003

The first recorded knockout mouse was

Knockout mice are used to study about what happen in an organism when a particular gene is
absent. Studying knockout mice can provide information about how the knocked-out gene
normally functions, including the gene's biochemical, developmental, physical, and
behavioral roles.
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.

STRAINS
There are several thousand different strains of knockout mice. Many 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 tumors by
arresting cell division and/or inducing apoptosis. Humans born with mutations that deactivate
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.
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 the gene inoperable.
Usually, the new sequence is also given a marker gene. in addition, a second gene, such
as herpes tk+, is also included in the construct in order to accomplish a complete
selection.
2. Embryonic stem cell is 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. By the natural process of homologous recombination 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 knocked-out embryonic 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.

5 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 do not retain
any grey mouse DNA and are not chimeras, however they are still heterozygous

6 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
unaltered gene (i.e. they are homozygous for that allele)

.
LIMITATIONS
The National Institutes of Health discusses some important limitations of this technique.
While knockout mouse 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. This problem is often
overcome through the use of conditional mutations. 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 tumors in a particular set of tissues. However, when the p53
gene is knocked out in mice, the animals develop tumors in a different array.
Knock-out v\s Knock-In Mice?
KNOCK-IN and KNOCK –OUT MICE are both genetically altered mice that assist
researchers in understanding the genetic functions of the human body at a
greater level of detail. While doing genetic research directly on humans and
modifying genes in human tissues is still not an ethical, viable option, genetically
modified mice can present researchers with a window of opportunity that other
models may not offer. This is due to the fact that the mouse genome – albeit far
simpler – is extremely similar to the human genome. It contains many of the
same genes that scientists would need to knock out or knock in, depending on
their research goals. Despite both methods being used for genetic research,
that’s pretty much where the similarities between knock-in and knockout mice
end. In fact, the processes of generating both types of model are quite different.
Moreover, the entire approach to the study of knockout mice is very different
compared to that of knock-in mice.

The most important difference between the two types of models is that, in the
case of knockout mice, a gene is targeted and inactivated, or “knocked out.” On
the other hand, generating knock-in mice involves the opposite technique:
altering the mouse’s genetic sequence in order to add foreign genetic material in
the form of a new gene housed by the specific locus targeted by the researcher.
Knockout mice are far older and more vastly researched when compared to
knock-in models. The first knockout mouse was generated in 1989, while
generating knock-in mice is a process that has only been perfected during the
past few years. However, this technique has been growing in popularity at an
accelerated rate, as the methods of obtaining knock-in mice continue to increase
in number.

Knock-in and knockout technologies are very different. While both can be used
on laboratory mice to facilitate genetic research without having to experiment on
human subjects, the technology and the specific methods used to delete or
inactivate a portion of the DNA sequence are distinct and recognized as such by
researchers who are familiar with both gene knockouts and knock-ins. Knock-ins
create a one-for-one substitution of the DNA sequence within the genome. In
particular, knock-in technology is focused on a specific locus of the sequence,
compared to how knockout mouse generating technologies and methods focus
on engineering an entirely new DNA sequence, which is very similar to the
original one, but made to ensure that the gene becomes inoperable.