Gene knockout technology involves replacing or disrupting a gene with artificial DNA to study gene function. The first recorded knockout mouse was created in 1989. Knockout mice and microorganisms are common tools used to study gene function in the laboratory. The process involves isolating a gene from a mouse library, engineering a new DNA sequence with a marker gene, introducing the sequence into stem cells via electroporation, and implanting the stem cells into mouse blastocysts to generate knockout mice.

2. KNOCKED OUT AN EXISTING GENE BY
REPLACING IT OR DISRUPTING IT WITH AN
ARTIFICIAL PIECE OF DNA.
KNOCKOUTS ARE USED TO STUDY THE FUNCTION OF
SPECIFIC GENES.
THE FIRST RECORDED KNOCKOUT MOUSE WAS
CREATED BY MARIO R. CAPECCHI, MARTIN
EVANS AND OLIVER SMITHIES IN 1989
GENE KNOCK OUT
TECHNOLOGY

3. A laboratory mouse in which a gene affecting hair
growth has been knocked out (left), is shown next
to a normal lab mouse

4. KNOCKOUT MICE ARE A COMMON TOOL FOR
STUDYING GENES IN THE LABORATORY.
MICROORGANISMS ARE ALSO USED FOR
STUDYING GENE FUNCTION.
THE KNOCKOUT CAN BE LETHAL.
ANIMAL MODELS

5. WITH THE USE OF RECOMBINANT DNA TECHNOLOGY CLONED GENES ARE
MODIFIED IN SUCH A WAY, THAT THEY ARE NO LONGER FUNCTIONAL,
CLONED GENES MODIFIED IN THIS WAY ARE KNOWN AS ‘KNOCK OUT
CONSTRUCT’, WHEN HOMOLOGOUS RECOMBINATION OCCURS WITH A
KNOCK OUT CONSTRUCT.THE NON-FUNCTIONAL KNOCKOUT ALLELE
REPLACE THE ENDOGENOUS MILD TYPE ALLELE.
HOW IT WORKS?

6. THE GENE TO BE KNOCKED OUT IS
ISOLATED FROM A
MOUSE GENE LIBRARY
THEN A NEW DNA SEQUENCE IS
ENGINEERED
THE NEW SEQUENCE IS ALSO GIVEN
A MARKER
GENE.
STEM CELLS ARE ISOLATED FROM A
MOUSE BLASTOCYST.
PROCEDURE
,

7. • The new sequence is introduced into the stem
cells by electroporation.
The stem cells that incorporated the knocked-out
gene are isolated.
The knocked-out stem cells are inserted into a
mouse blastocyst.
These blastocysts are then implanted into
the uterus of female mice

8. • The newborn mice will therefore
be chimeras.
These chimera mice are crossbred with
others of the wild type.
The mice produced are not chimeras, but
they are still heterozygous.
When these heterozygous offspring are
interbred, some of their offspring will inherit
the knocked-out gene from both parents;

9. KNOCKOUT MOUSE GENE IN ES CELLS

10. APPLICATIONS
To determine the function of gene products.
To create mouse modal of human genetic diseases.
To characterize genetic regulatory regions.

11. CYSTIC FIBROSIS
Cystic fibrosis is a genetic disorder that perticularly
affects the lungs and diagestive system.
CF is inherited disease.
The inherited CF gene directs the body’s epithelial
cells to produce a defective form of a protein called
CFTR(cystic fibrosis transmembrane conductance
regulator) found in cell line of lungs,digestive
tracts,sweat gland,and genitourinary system.

12. CF is inherited disease.
The inherited CF gene directs the body’s epithelial cells to
produce a defective form of a protein called CFTR(cystic
fibrosis transmembrane conductance regulator) found in
cell line of lungs,digestive tracts,sweat gland,and
genitourinary system.
When the CFTR protein is defective,epithelial cells can’t
regulate the way chloride (part of salt called sodium
chloride) passes across cell membrane.

13. • This disrupts the essential balance of salt and water
needed to maintain a normal thin coating of fluid and
mucus insight the lungs, pancreas and passege ways in
other organs.The mucus become thick, sticky and hard
to move.
Normally, mucus in lungs traps germs,which are then
cleared out of the lung. But, in CF, the thick,sticky
mucus and germs it has trapped remain in lungs, which
become infected.

14. • The human Cystic Fibrosis gene is identified and cloned
in a phage vector.
ES cells are recoverd from mouse blastocyst.
Transfection of ES cells with CFTR knockout construct.
Animals homozygous for the CFTR knock out allele
display a mutant phenotype that is very similar to that
expressed by humans suffering from Cystic Fibrosis.
Thus ,in developing drugs to alleviate CF system in
human,pharmaceutical researches can first test new
products in mice to determine their efficiency.

15. LIMITATIONS
Genetically altered embryos cannot grow into
adult mice.
The lack of adult mice limits studies to embryonic
development.
 The gene may serve a different function in adults
than in developing embryos.