describing the principles of gene knockout and its application

1. GENE KNOCKOUT
BY SAMUEL KWATIA
M.Sc Biotechnology. KNUST 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
• Reverse genetics is used to determine the knockout organism and normal organism.
• 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
• Researchers who developed tchreea tteiocnh noofl okgnyo cfkoor utth em ice 2w0o0n7 .Nobel Prize in the year
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" .
• Mario . R Capecchi
gene knockout
• Sir Martin J. Evans
cultivation of ES 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).
• gCeonnes.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
• lGibernaery .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
• flanked by two arms of homologous sequence
• to enrich recombination events.
• expression cassettes encoding antibiotic
resistance genes.
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
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. 2009 September ;
CHAPTER: Unit–19.1217. doi:10.1002/0471143030.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.
11

12. ES CELL TRANSFECTION
• Stem cells combined with the new sequence through electroporation and cultured.
• random integration occurs
• hoof mneowlo ggoeunse retoc ormepbliancaeti oonld o occnuer.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.
12

13. RECOMBINATION
Homologous recombination Random integration
13

14. REGENERATION
• Positive stem cells are incorporated into the blastocyst
cells of another mouse.
• The blastocysts contain two types of stem cells (chimera): the original ones (grey mouse), and the
newly engineered ones (white mouse)
• These blastocysts are then implanted into the uterus of female mice, to complete the pregnancy.
• 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. 14

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)
15

16. OVERALL PROCESS
16

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

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
disease, arthritis, Parkinson’s disease 18

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.
21

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
22

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
• The Nobel Prize in Physiology or Medicine
2007514551 http://www.genome.gov/12^ 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: 541-545.
•a b genome.gov | Background on Mouse as a Model
Organism
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)
26

27. 27
THANKS FOR YOUR AUDIENCE