Knockout mice are genetically engineered mice where one or more genes have been inactivated through gene knockout. They are important animal models for studying the role of genes with unknown functions. By causing a specific gene to be inactive in mice and observing differences in behavior or health, researchers can infer the probable function of that gene. Transgenic mice have foreign or modified genes added, which are then integrated randomly into the mouse genome, allowing the study of these additional genes. Both knockout and transgenic mice are useful models for studying human genetic diseases.

1. KNOCK OUT MICE
DR. KHUSHBOO BHOJWANI
BDS, MSC PHARMACEUTICAL MEDICINE

2. KNOCKOUT MICE
• A knockout mouse is a genetically engineered mouse in
which one or more genes have been turned off through a
gene knockout
• Important animal models for studying the role of genes
which have been sequenced, but have unknown functions
• By causing a specific gene to be inactive in the mouse,
and observing any differences from normal behaviour or
condition, researchers can infer its probable function.

3. TRANSGENICS
 This technique permits the introduction of foreign genes or
altered forms of an endogenous gene into an organism
 Mostly, this technique does not result in replacement of
the endogenous gene, but rather the integration of
additional copies of it
 The introduced gene is called transgene and the organism
carrying it is referred to as transgenic

4. KNOCKOUT VS. TRANSGENIC
MOUSE?
Knockout:
-gene inactivated
-homologous recombination
Transgenic:
- gene(s) added can be:
- a foreign gene
- extra copies of endogenous gene
- mutated endogenous gene

5. TRANSFERRING DNA INTO
EUKARYOTIC CELLS
• Production of both knockout and transgenic animals
requires the transfer of DNA into eukaryotic cells.
• Calcium precipitation
• Liposome delivery
• Microinjection
• Electroporation
•

6. • Calcium phosphate precipitates of
DNA form when DNA is mixed with
calcium chloride. When these DNA
precipitates are added to animal
cells growing in culture, the
precipitated DNA can be taken up by
the cells, again transferred to the
nucleus and expressed
• Liposomes are artificial membranes
that can be formed in a test tube.
DNA can be mixed with the
liposome preparation under the
appropriate conditions. This results
in the encapsulation of DNA into
synthetic lipid membranes. When
this membrane fuses with the cell
plasmamembrane, DNA is released
into the cell and somehow ends up
in the nucleus.

7. DNA MICROINJECTION
DNA can also be
injected directly into
the nuclei of both
cultured cells and
developing embryo

8. ELECTROPORATION
• Cells are subjected to a brief electric
shock of several thousand volts and
become transiently permeable to DNA.
Presumably the shock briefly opens holes
in the cell membrane allowing the DNA to
enter the cells before the holes reseal

9. DNA INCORPORATION IN THE
CELL
 Once the foreign DNA is inside the host cell, enzymes that
function normally in DNA repair and recombination join the
fragments of foreign DNA into the host cells chromosome
 The new fragment can either replace an endogenous gene-
homologous recombination or it can remain as an
independent extrachromosomal DNA molecule referred to
as an episome

10. IDENTIFICATION OF
TRANSGENIC CELLS
• Since only a relatively small fraction of cells take up DNA,
a selective technique must be available to identify the
transgenic cells
• In most cases the exogenous DNA includes two additional
genes
• The small fraction of cells in which homologous
recombination takes place can be identified by a
combination of positive and negative selection

11. POSITIVE AND NEGATIVE
SELECTION
 Positive Selection- One of the additional genes (neoR) confers
neomycin resistance; it permits positive selection of cells in
which either homologous (specific) or non-homologous
(random) recombination has occurred
 Negative selection- The second gene, thymidine kinase gene
from Herpes Simplex Virus (tkHSV) confers sensitivity to
gancyclovir(a cytotoxic nucleotide analog). This gene permits
negative selection of ES cells in which non-homologous
recombination has occurred
 Only ES cells that undergo homologous recombination (i.e.
gene-targeted specific insertion of the DNA construct) can
survive this selection scheme

13. POSITIVE AND NEGATIVE
SELECTION OF RECOMBINANT ES
CELLS
Recombinats with
random insertion Nonrecombinat cell
Recombinats with gene-
targeted insertion
Treat with neomycin
(positive selection)
Treat with gancyclovir
(negative selection)

14. POSITIVE SELECTION
MARKERS
To enrich recombination events
Expression cassettes encoding antibiotic resistance genes

15. NEGATIVE SELECTION
MARKERS
Used to enrich for homologous recombination events over
random insertions.
Use of Herpes Simplex Virus (HSV) Thymidine Kinase (TK)
gene coupled with gancyclovir treatment

16. HOMOLOGOUS
RECOMBINANT

18. KNOCKOUT MICE
Gene knockout is a technique for selectively inactivating a
gene by replacing it with a mutant allele in an otherwise
normal organism (mice)
Knockout mice are a useful model system for studying
certain human genetic diseases.

19. MAKING KNOCKOUT
MICE
 Mutant alleles are introduced by
homologous recombination into
Embryonic Stem cells
 ES cells containing the knockout
mutation are introduced into early
mouse embryos. The resultant mice
will be chimeras containing tissues
derived from both the transplanted
ES cells and host cells. These cells
can contribute to both germ cell and
somatic cell population
 Chimeric mice are mated to assess
whether the mutation is incorporated
into the germline
 Chimeric mice each heterozygous for
the knockout mutation are mated to
produce homozygous knockout mice

20. PROCEDURE FOR
MAKING MIXED
GENOTYPE BLASTOCYST

21. DRAWBACKS OF KNOCKOUT
MICE
• About 15% of gene knockouts are developmentally lethal
and therefore cannot grow into adult mice. Thus it
becomes difficult to determine the gene function in
adults.
• Many genes that participate in interesting gene pathways
are essential for either mouse development, viability or
fertility. Therefore , a traditional knock out of the gene can
never lead to the establishment of knockout mouse strain
for analysis

22. MICE - MODELS OF
HUMAN DISEASES
 Although the human is the mammal we are generally most
interested in learning more about, it is also the one animal
we cannot use for genetic experiments for obvious ethical
reasons
 Mice naturally develop conditions that mimic human
disease, such as cardiovascular disease, cancer and
diabetes
 Mouse are a favorite model for human disease because it
has a relatively low cost of maintenance and a generation
time that measures only nine weeks

23.  Developments in molecular biology and stem cell biology
have allowed researchers to create custom-made mice
through gene targeting in mouse embryonic stem (ES)
cells
 Certain diseases that afflict only humans, such as cystic
fibrosis and Alzheimer's can also be induced by
manipulating the mouse genome and environment

24. KNOCKOUT MICE TO STUDY
GENETIC DISEASES
Knockout mice make good model systems for investigating
the nature of genetic diseases and the efficacy of different
types of treatment and for developing effective gene
therapies to cure these often devastating diseases
For instance, the knockout mice for CFTR gene show
symptoms similar to those of humans with cystic fibrosis

25. RESEARCH USING KNOCKOUT
MICE
Examples of research in which knockout mice have been useful
include studying and modelling different kinds of
-obesity
-heart disease
-diabetes
-arthritis
-Parkinson’s disease
Knockout mice also offer a biological and scientific context in
which drugs and other therapies can be developed and tested.

26. TRANSGENIC ANIMALS
• Transgenic animals carry cloned genes that have integrated
randomly into the host genome
• Transgenic technology has numerous experimental application
and potential therapeutic value
• The frequency of random integration of exogenous DNA into
mouse genome at non-homologous sites is very high, therefore,
the production of transgenic mice is a highly efficient and
straightforward process

27. •Foreign DNA containing a gene of interest is injected into one of the two
pronuclei (the male and female nuclei contributed by the parent) of a
fertilized mouse egg before they fuse
•The injected DNA has a good likelihood of being randomly integrated into
the chromosome of the diploid zygote

28.  Injected eggs are then
transferred to foster
mothers in which normal
cell growth and
differentiation occurs
 About 10-30% of progeny
will contain the foreign
DNA in equal amounts in
all tissues, including the
germ cells
 Immediate breeding and
backcrossing of these
mice can produce pure
transgenic strains
homozygous for the
transgene

29. TRANSGENICS AND GENE
THERAPY
 Once a gene mutation is identified to be the cause of a
disease, the next step is to cure the disease by introducing
normal genes( transgene) into affected individual
 In experimental animals, some genetic disorders have
been cured by gene therapy, but in humans, numerous
technical issues need to be resolved before it can be
widely used
a) how to reliably and safely introduce various genes
into human cells
b) tissue/ cell specific introduction of genes
c) large size of genes
d)how to address the ethical issues