A gene knockout is a genetic technique in which one of an organism's genes is made inoperative ("knocked out" of the organism). However, gene knockout can also refer to the gene that is knocked out or the organism that carries the gene knockout. Knockout organisms or simply knockouts are used to study gene function, usually by investigating the effect of gene loss. Researchers draw inferences from the difference between the knockout organism and normal individuals.

1. GENE KNOCKOUT

2. ABSTRACT
A gene knockout is a genetic technique in which one of an organism's
genes is made inoperative ("knocked out" of the organism). However,
gene knockout can also refer to the gene that is knocked out or the
organism that carries the gene knockout. Knockout organisms or simply
knockouts are used to study gene function, usually by investigating the
effect of gene loss. Researchers draw inferences from the difference
between the knockout organism and normal individuals.

3. GENE KNOCKOUT
A gene knockout (abbreviation: KO) is a genetic technique in which one of
an organism's genes is made inoperative ("knocked out" of the organism).
However, KO can also refer to the gene that is knocked out or the organism
that carries the gene knockout. Knockout organisms or simply knockouts
are used to study gene function, usually by investigating the effect of gene
loss. Researchers draw inferences from the difference between the
knockout organism and normal individuals.
The KO technique is essentially the opposite of a gene knockin. Knocking
out two genes simultaneously in an organism is known as a double
knockout (DKO). Similarly the terms triple knockout (TKO) and quadruple
knockouts (QKO) are used to describe three or four knocked out genes,
respectively.

4. Technologies for gene-knockout
The best approach to produce a gene knockout is homologous recombination and
through gene knockout methods a single gene gets deleted without effecting the all
other genes in an organism. With the help of the gene knockout the organism where
the gene of interest becomes inoperative is known as knockout organism. When
more than one gene is get knocked out in an organism then is called double knock out
or DKO, triple knockout or TKO and quadrule knockouts or QKO depending on the
number of genes.

5. Gene technology procedure
Gene knockout is carried out together with elements such as plasmid, DNA construct
or bacterial artificial chromosome.
Gene knock out procedure often generate transgene animals where the target gene
has been altered. To produce transgenic animals, embryonic stem cells or ES cells get
genetically modified and in following step the transformed ES cells are placed in early
embryos. The transformed animals thus produce has the ability to carry forward the
transformed gene in following generations

6. Equipment
1) Thermo cycler for PCR
2) Agarose gel apparatus
3) Power supply
4) Spectrophotometer to read
DNA concentration at
260nm and cell density at
600nm
5) Electroporator
6) Constant temperature
bacterial incubator set at
30°-32°C. Should contain
a roller for liquid culture
tubes and shelves for petri
plates 32° and 42°C
shaking (200rpm) water
baths (42°C cannot be an
air shaker)
7) Low-speed centrifuge with
Sorvall SA-600 rotor (or
equivalent) at 4°C
8) Refrigerated
microcentrifuge at 4°C
9) Gel imaging system
10) Insulated ice bucket
11) Sterile 35 to 50 ml plastic
centrifuge tubes
12) Sterile 50 and 125 ml (or
250) Erlenmeyer flasks,
preferably baffled
13) Micropipettors
14) Sterile, aerosol-resistant
pipettor tips
15) Pipettes of various sizes
16) PCR tubes (0.2 ml flat cap
tubes)
17) 5 ml microfuge tubes
18) Sterile glass culture tubes
with stainless steel closures
for culturing bacteria
19) Spectrophotometer
cuvettes
20) Electrotransformation
cuvettes with 0.1cm gap
(pre-chilled)
21) Petri Plates – 100 x 15mm
22) Optional but highly
recommended: DNA
analysis software

7. Different Methods for gene
knockout
Homologous Recombination- homologous recombination is the
conventional method for gene knockout and widely used in genome
engineering. This method comprises of nucleotide exchange between
DNA sequences which are either similar or identical. Homologous
recombination method includes a DNA construct with the mutation of
choice and a drug resistance cassette to be interchanged in place of
knockout gene. Additionally, the construct also includes a homologous
region of nearly 2Kb with the target gene. Microinjection or
electroporation are the most common methods which are next applied
to transfer the construct into desired organism.

8. Homologous recombination
Traditionally, homologous recombination was the main method for
causing a gene knockout. This method involves creating a DNA
construct containing the desired mutation. For knockout purposes, this
typically involves a drug resistance marker in place of the desired
knockout gene. The construct will also contain a minimum of 2kb of
homology to the target sequence. The construct can be delivered to
stem cells either through microinjection or electroporation. This method
then relies on the cell's own repair mechanisms to recombine the DNA
construct into the existing DNA. This results in the sequence of the gene
being altered, and most cases the gene will be translated into a
nonfunctional protein, if it is translated at all.

9. Contd.
Site-Specific Nucleases- There are namely three methods, zinc fingers,
TALENS and CRISPER which is known to introduce double stranded
breaks in DNA. Following DNA damage, the cells own repair mechanism
get functional through non-homologous end joining (NHEJ), to ligate two
open ends. The repair mechanism being finished imperfectly generates
insertion or deletion mutation which results in frame shift mutation.
Following the mutation the gene produces non-functional protein and
generates a knockout for the gene of interest.

10. Zinc-Fingers
Applications-
Zinc finger nucleases (ZFNs) are used for creating complete knockout in
several cell lines.
They can be used in cell based screening methods by generating knock-
in cells lines where endogenous target genes can be tagged with
promoter, reporter or fusion tag proteins.
They are also useful for generating cell lines to produce specific protein
or antibodies in higher amount.

11. TALENS
Applications-
TALENs are used widely for plant genome modification .
They are useful for the production of biofuels.
They have been used to generate knockout in organisms such as
zebrafish, rat , mice or c.elegance
It also been used to treat genetic diseases such as xeroderma
pigmentation ot sickle cell .

12. CRISPER/Cas9
Applications-
CRISPER/Cas9 gene editing system is useful for Homology-directed
repair (HDR) mechanism.
They can be used for gene silencing
They are functional for transiently activate endogenous genes.
It is useful for DNA free gene editing methods.
They are used for transiently silenced expression of genes.
Preparation of transgenic animals as well as embryonic stem cells.

13. USES
Knockouts are primarily used to understand the role of a specific gene or DNA region
by comparing the knockout organism to a wildtype with a similar genetic background.
Knockout organisms are also used as screening tools in the development of drugs, to
target specific biological processes or deficiencies by using a specific knockout, or to
understand the mechanism of action of a drug by using a library of knockout
organisms spanning the entire genome, such as in Saccharomyces cerevisiae.

14. REFERENCE
1) National Programme for Technology Enhanced Learning, a project
funded by MHRD, Govt. of India.
2) Molecular Biology Of The Gene by James. D. Watson, PEARSON
Publication.
3) https://en.wikipedia.org/wiki/Gene_knockout
4) Biotechnology by P.K.Gupta, Rastogi Publication.
5) Biotechnology by B.D.Singh, Kalpana publication.