Gene knockout in mice

knockout organism carries genes that have made inoperative

1. GENE KNOCKOUT

2. •A gene knockout is genetically engineered organism that
carries one or more gene in its chromosome that have
made inoperative
•Reverse genetic tools
•Used to study the function of specific gene
•It is based on gene targeting, a technique use
homologues recombination to modify genome of living
organism
•Other forms of gene disruption are
1.Gene knockdown
2.Gene knockin
3.Gene knockon
4.Gene knockabout

3. •Knockouts are
2 genes = double knockouts
3 genes = triple knockouts
4 genes = quadruple knockouts

• Mario R.Capecchi, Sir Martin J. Evans and OliverSmithies
created in andwon nobel prize 2007(1989) "for their
discoveries of principles for introducing specific gene
modifications in mice by the use of embryonic stem cells" .

•Gene function cannot be determined by amino acid motifs
•Half of mouse genome have been knocked out

4. MODELS
Animals (rats)
(yeast)
(Mycoplasma
genitalium)
Plants
(Arabidopsis
thaliana )

7. •Embryonic Stem Cell Culture
•ES are undifferentiated cells isolated from inner cell mass of the
blastocyst
•Pluripotent, including germ cell
•ES cells in culture grown well and remain undifferentiated
•Cytokine leukaemia inhibitory factor (LIF) to ensure that ES cells
do not differentiate in vitro, thus ES cells are grown on feeder
layer fibroblasts
•procedure will vary depending on the strain
•Generally cells derived from strain 129 and p53 are used. This
specific strain is common to backcross the offspring to other
strains.

8. •More Advanced type of Knockouts
• Conditional knockouts or tissue specific gene targeting.

• The goal of conventional knockout technology is to knockout both
alleles so that the gene is entirely absent from all cells.

• The purpose of conditional knockouts is to delete a gene in a particular
organ, cell type or stage of development.

• Conditional knockout consists of several merits over conventional
knockouts i.e.,
a) Longer survival than traditional one.
b) It is more precise.

9. A laboratory mouse in which a gene affecting hair
growth has been knocked out (left), is shown next
to a normal lab mouse
A knockout mouse (left) that is a model of obesity,
compared with a normal mouse.

10. Normal knockout
GDF8 (Myostatin) knockout mouse More than twice the muscle mass of a wildtype mouse
FGF5 knockout mouse has long, angora-like hair

11. USES
•Knockout mice have been useful in studying and modeling different kinds of
cancer, obesity, heart disease, diabetes, arthritis, anxiety, aging and Parkinson disease.

•Knockout mice also offer a biological context in which drugs and other therapies can be
developed and tested.

12. Limitation
•Some loci are very difficult to knock out. Reasons might be the presence of repetitive sequences,
extensive DNA methylation, or heterochromatin

•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.

•For example, when the p53 gene is knocked out in mice, the animals develop tumours in a different
array of tissues.

•About 15 percent of gene knockouts are developmentally lethal (genetically altered embryos
cannot grow into adult mice.)

•The lack of adult mice limits studies to embryonic development and often makes it more difficult to
determine a gene's function in relation to human health.

•The gene may serve a different function in adults than in developing embryos.

• No efficient methods of gene knockout have yet been developed for use in plants, because
Insufficient frequency or efficiency of homologous recombination.