This ppt focus on One major Molecular Biology Technique : Gene Knockout .

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2. GENE KNOCKOUT
Presented By
Dr. Kazi Shams Al Arefin
DVM, MS(MICROBIOLOGY)
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3. Knockout means
The gene knockout is a molecular genetic technique used to study the function
of a gene, abbreviated as KO.
 Use of genetic engineering to inactivate or remove one or more specific genes
from an organism.
 Knockout can be produced by removing the gene or inducing a mutation that
disable it’s function.
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4. This technology helps one to study the function of gene by observing what
happens when a gene is absent or when mutant gene is expressed instead of
normal one.
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).
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5. Pioneer of KOT
The Nobel Prize in Physiology or Medicine 2007 was awarded jointly to Mario R.
Capecchi, Sir Martin J. Evans and Oliver Smithies "for their discoveries of
principles for introducing specific gene modifications in mice by the use of
embryonic stem cells“.
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6. Process of gene knockout
1.Selecting a gene for knockout
2. Construction of vector
Vector is a vehicle used to transfer our gene of interest or any other DNA sequence
to our target cells, a plasmid is generally used for it.
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General structure of a plasmid used in genetic engineering experiments.

8. Suppose we have introduced a frameshift mutation into our DNA sequence,
which inhibits protein formation.
Now for the safer side, to validate our results a marker DNA sequence is also
introduced in it, generally, an antibiotic resistance gene is used for it.
• A marker gene(AMRG) is inserted only to make the insert detectable for
reporting the results, it works as a reporter.”
• NeoR gene- neomycin resistance gene is one of the popular reporter or marker
sequence used in the gene knockout studies.
• Now plasmid is ready.
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9. 3. Insertion into ES cells:
• Plasmid is inserted into the ES cells such as electroporation, sonication or
microinjection methods.
• If it finds the target gene, recombination will occur and a mutation is
inserted into the target gene.
• Now our transformed cells are grown into the neomycin-containing media
so that the cells containing the NeoR gene can grow.
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10. 4.Confirming the insert:
• When we grow our cells under in vitro conditions, it is possible that some cells
may be transformed or some cells may not.
• Now using the polymerase chain reaction, the insert can be confirmed.
5.Injecting into the embryo:
• Now pick transformed cells and insert them into the developing embryo of our
model organism.
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11. 6.Breeding:
• Now the embryo of model organism has two types of cell population, one wild
type and one altered (transformed) cell, this animal is called chimeric(GM)
• Then, we breed it with the normal animal which produces offspring of two
different genotypes: one with homozygous normal or another animal with
homozygous altered genotype (and heterozygous as well).
• Now our gene knockout animal is constructed, scientists can examine it for
measuring a different parameters related to our gene of interest, using the PCR
amplification method, the results of gene knockout can be validated.
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13. Why knockout mice
• We are using the mice in the genetic engineering studies and knockout
studies because of the similarities between the genes of humans and mice.
• 99% of human and mice genes are similar, thus instead of using human
embryos directly for the experiment, using mice is a wise decision.
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14. Methods
1.Gene silencing
2.Conditional knockout
3.Homologous recombination
4.Gene editing
5.Knockout by mutation
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15. Homologous recombination
• Exchange of the nucleic acid between identical or homologous sequences occurs through
homologous recombination.
• Insert the gene of interest, in place of our target gene.
• Identical DNA sequences of up to 2Kb are inserted in the vector along with the antibiotic
resistance gene and incorporated into the target genome using ,electroporation,
microinjection.
• Once the vector is inserted into the cell it recombined with the target DNA sequence, our
DNA of interest with the antibiotic resistance gene inserted into the target genome.
• This method is one of the simplest and most effective methods used for long, however, the
efficiency is very low.
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17. Application
• Study the function of a particular gene.
• It also enables scientists to monitor and control the effect of a gene.
• Constructing genetically modified organisms such as GM plants, GM bacteria and
GM animals.
• study the effect and contribution of a particular gene and its role in the
development of a disease.
• It is likewise employed in drug discovery: using gene knockout like genetic
engineering tools, drug screening can be done.
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18. Overview whole process
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Step 1. isolate a blastocyst-stage developing embryo. This embryo came from a type of gray-furred mouse.
Step 2. Remove the gray-fur blastocyst's embryonic stem cells. In tissue culture, grow stem cells.
Step 3. Use a homologous recombination construct to transfect stem cells. Stem cells are grown with neomycin and ganciclovir to
select for homologous recombination.

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Step 4. Take homologous recombined stem cells out of the petri dish and insert them into an unfurred fresh blastocyst.
Step 5. Implant various chimeric blastocysts into pseudo-pregnant, white fur mouse.
Step 6. Mother will give birth to a range of mice. Some will be normal white fur mice but others will be chimeric mice.
Chimeric mice have many of their cells from the original white fur blastocyst but some of their cells will be derived from
recombinant stem cells.

20. Advantages
• To study effect of gene product
• Help in study the function of gene.
• Biological pathways .
• Test the beneficial effect of drug and gene therapy.
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21. Disadvantages
• 15% show development lethality.
• Very expensive( 3000 to 30000)dollar.
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22. Knockout vs Knockdown Vs Knock in
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23. drawbacks
• Gene knockout models are much more expensive than standard transgene
procedure because of the extensive embryonic stem cell culturing and analysis.
• Other limitations are the possibility of incomplete inactivation, and disruption of
overlapping or adjacent genes.
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