1. GENE KNOCKOUT 1

2. INTRODUCTION
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)
The technology of gene knockout is based on gene targeting, a useful technique that utilizes homologous recombination to modify the genome of a living organism.
The term also refers to the process of creating such
an organism, as in "knocking out" a gene. 2

3. INTRO. CONT’D
Knockouts are basically used to study the function of specific genes
Roregvaenrissemgaenndetincosrmisalusoergdantoismde.termine the knockout
Other forms of gene disruption
gene knockdown… reduce expression of the gene
knock-in… replace one allele (e.g., wild type) with
another (e.g. a specific mutation) 3

4. INTRO. CONT’D
A conditional knockout allows gene deletion in a tissue specific manner.
Simultaneously knocking out
2 genes →
double knock out
3 genes
triple knockout
4 genes
quadruple knockout 4

5. HISTORY discoveries of principles for•
Mario . R Capecchi
gene knockout
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cwroenatiNonoboefl kPnroizcekoinut thmeicyeear
The Nobel Prize in Physiology or Medicine was •
awarded jointly to Mario R.
Sir Martin J. Evans
cultivation of ES cells
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". •
Oliver Smithies
gene targeting 5

6. BASIC METHOD FOR GENE KNOCK
OUT
Gene knockout is accomplished by a combination of techniques. Beginning from the test tube with plasmid, a bacterial artificial chromosome or other DNA construct, and then proceeding to cell culture.
Genetically, individual cells are transformed with a construct. ( knockout in multicellular organisms use Stem cell from nascent embryo).
Cgeonnes.truct is engineered to recombine with the target 6

7. METHOD CONT’D
With its sequence interrupted, the altered gene in most cases will be translated into a non – functional protein.
Recombination is a rare event → therefore foreign sequence chosen for insertion usually is a reporter for easy selection of recombinants. 7

8. KNOCKOUT MOUSE
Glibernaery.to be knocked out is isolated from mouse gene
Generation of targeting vector
contains pieces of DNA that are homologous to target gene, just inoperative.
positive and negative selection markers / cassettes ( SC)
neomycin phosphotransferase (neor) gene and HSV thymidine kinase (HSV-tk) gene respectively 8

9. KNOCKOUT MOUSE CONT’D Positive negative outside one homologous arm
used to enrich for homologous recombination events over random insertions.
Use of Herpes Simplex Virus (HSV) Thymidine Kinase (TK) gene coupled with gancyclovir
flanked by two arms of homologous sequence
to enrich recombination events.
expression cassettes encoding antibiotic
resistance genes.
treatment 9

10. KNOCKOUT MOUSE CONT’D
Two homology arms flank a positive drug selection marker (neor). A negative selection marker (HSV-tk) is placed adjacent to one of the targeting arms. A unique restriction enzyme site is located between the vector backbone and the homology arm. When linearized for gene targeting, the vector backbone will then protect the HSV-tk from nucleases.
Overview: Generation of Gene Knockout Mice, Bradford Hall1, Advait Limaye1, and Ashok B Kulkarni1,1 Curr Protoc Cell Biol September ; CHAPTER: Unit– doi: / cb1912s44. 10

11. EMBRYONIC STEM( ES) CELL ISOLATION
Embryonic stem (ES) cells are undifferentiated cells
isolated from the inner cell mass of a blastocyst
(Evans and Kaufman, 1981).
Are pluripotent
Most importantly the three germ layers – ectoderm, endoderm and mesoderm.
Replicate indefinitely.

12. ES CELL TRANSFECTION
Selteecmtrocpeollrsaticoonmabnindedcuwltuithredth. e new sequence through
random integration occurs
hofomneowloggoeunseretocormepbliancaetioonldooccnuer.s → incorporation
The antibiotic genes will aid in selection of mutants.
Discrete colonies are identified and picked for
screening of positive clones.
PCR
Southern blotting and DNA sequencing.

13. RECOMBINATION
Homologous recombination
Random integration

14. REGENERATION
Positive stem cells are incorporated into the blastocyst cells of another mouse.
T(chheimbelaras)t:octhyestsorcigoinntaalinontweso (tgyrpeeys mofoussteem), acnedllsthe newly engineered ones (white mouse)
Tofhefesme ablelasmtoiccey,sttso acroemtphleenteimthpelapnrteedgnianntocyt.he uterus
The newborn mice will therefore be chimeras: parts of
tphaeritrs broedsuielts frreosmultthferomengthineeeorreigdinsatlemstecmellcse. lls, other

15. REGENERATION CONT’D Their furs will show patches of white and grey
New-born mice are only useful if the newly engineered sequence was incorporated into the germ cells (egg or sperm cells)

16. OVERALL PROCESS

17. A. chimeric. mouse. gene targeted. for. the. agouti coat. color. gene,
A chimeric mouse gene targeted for the agouti coat color gene, with its offspring

18. APPLICATIONS OF GENE KNOCKOUT
Allows the test of specific functions of particular
genes and to observe the processes that these particular genes could regulate.
Enables us to monitor the effects a particular gene.
Biomedical research-understanding how a certain gene contributes to a particular disease, researchers can then take the knowledge a step further and look for drugs that act on that gene. E.g. obesity, heart 18
disease, arthritis, Parkinson’s disease

19. May lead to the discovery of the next generation of therapies for curing numerous diseases based on novel targets from the human genome. 19

20. CONCERNS
Many knockout mice die while they are still embryos before the researcher has a chance to use the model for experimentation.
There is mostly increased cost in caring for genetically altered organism
Some religious organizations have objections to the use of embryonic stem cells. Many other groups
disagree with their use as well 20

21. CONCERNS CONT’D
The gene may serve a different function in adults than in developing embryo
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. E.g. mutations in the p53 gene associated with cancers and tumours.

22. GENE KNOCKOUT IN PLANTS
Use of gene targeting in plants has proven very difficult.
No efficient methods of gene knockout have yet been developed for use in plants.
Insufficient frequency or efficiency of homologous recombination.
This is overcome by any of these ways

23. GENE KNOCKOUT IN PLANTS
CONT’D
Gene targeting with transposons and bacterial recombination systems.
These consist of recognition sequences and an enzyme( Transposase or recombinase) that cut DNA segments out of the genome and reintegrate at the recognition sequences and reintegrate them at another site 23

24. GENE KNOCKOUT IN PLANTS
CONT’D
Gene targeting through stimulation of the cell’s own recombination processes.
In this method, a special DNA-cutting enzyme (“I-SceI” restriction enzyme) is used to cut the DNA strand at two sites in the plant genome. It is then possible at these sites to carry out recombination processes and so to achieve a targeted exchange of homologous sequence segments. 24

25. REFERENCES a b genome.gov | Background on Mouse as a Model 25
The Nobel Prize in Physiology or Medicine
nature news,
19 May 2003.
Y Zan et al., Production of knockout rats using ENU
mutagenesis and a yeast-based screening assay, Nat. Biotechnol. (2003).
Alani, E., L. Cao, & N. Kleckner (1987). A method for
gene disruption that allows repeated use of
URA3selection in the construction of multiply disrupted
yeast strains. Genetics 116:
a b genome.gov | Background on Mouse as a Model 25

26. REFERENCES CONT’D
Evans, M. J. & Kaufman, M. H. Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156 (1981)

27. THANKS FOR YOUR AUDIENCE