Knockout mice are mice that have had a specific gene deleted or inactivated through genetic engineering. This allows researchers to study the function of genes by observing the effects of the gene's absence. Researchers use embryonic stem cells from mice to delete genes through a process called homologous recombination. Mice with the gene deletion are bred to generate strains of mice lacking the gene. Studying these knockout mice provides insights into the roles and functions of genes. Knockout mice are also useful models for studying human diseases and evaluating potential treatments.

1. KNOCKOUT MICE

2. KNOCK OUT MICE
• a mouse in which a gene has been deleted/mutated
(gene is inactivated)
• specific gene is targeted
• The loss of gene activity often causes changes in a
mouse's phenotype and thus provides valuable
information on the function of the gene.

3. Researcherswhodevelopedthetechnologyforthe
creationofknockoutmicewonNobelPrizeintheyear
2007
• 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".

5. 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

6. Why Make a KnockoutMouse?
• Determine the function of a particular gene/gene product.
• eliminate a specific protein
• ask: what can’t that animal do?

7. What Kinds of Questions ?
• Examine a protein’s role in a biological process
- protein X always increased during process Y
• Determine function of a newly identified gene
- genome sequencing projects (>30,000 genes)
- many of unknown function
- some info gained from comparisons

8. • Protein Families
- families of similar yet distinct proteins have the same
biochemical function
- all apparently do the same thing
- probably have different physiological roles
• Models for Human Diseases
- confirm mutation is responsible for disease
- studies not possible on humans
- experimental therapies

9. How to make a Knockout?
• Have ~ $40,000
• Alter gene of interest in Embryonic Stem (ES) Cells
• Use ES cells to make a mouse

10. GENERATIONOF KNOCKOUT MICE BY
HOMOLOGOUS RECOMBINATION
• Creating a knockout construct
• Introduce the knockout construct into mouse embryonic stem
cells (ES) in culture
• Screen ES cells and select those whose DNA includes the new
genes
• Implant selected cells into normal mouse embryos , making
“chimeras”
• Implant chimeric embryos in pseudopregnant females
• Females give birth to chimeric offsprings, which are
subsequently bred to verify transmission of the new
gene, producing a mutant mouse line

11. Knockoutconstruct:
• The gene to be knocked out is isolated from a mouse gene
library. Then a new DNA sequence is engineered which is very
similar to the original gene and its immediate neighbour
sequence, except that it is changed sufficiently to make the
gene inoperable. Usually, the new sequence is also given
a marker gene, a gene that normal mice don't have and that
confers resistance to a certain toxic agent or that produces an
observable change (e.g. colour or fluorescence).

12. EmbryonicStem(ES)Cells
• isolated from a pre-implantation embryos
- from inner cell mass of blastula stage embryo
• cells are undifferentiated
• cells are pluripotent
- able to differentiate into many different cell types in
embryo
- most importantly germ cells
• grown in culture
- need cells to divide but not differentiate
- longer time in culture = more differentation

14. The new DNA sequence is introduced into the
embryonic stem cells by electroporation. The
natural process of homologous recombination
occurs which means some of the
electroporated stem cells will incorporate the
new sequence with the knocked-out gene into
their chromosomes in place of the original
gene. The chances of a successful
recombination event are relatively low, so the
majority of altered cells will have the new
sequence in only one of the two relevant
chromosomes - they are said to
be heterozygous.

15. Gene Targeting Technology
• Cells take up exogenous DNA
- frequency is very low
- can be induced to higher frequency
- chemical, electrical, injection
• In Dividing Cells:
- some DNA incorporated into genome
1. Random integration
-rare event
2. Homologous Recombination
- even more rare

16. Random Integration:
• DNA incorporated anywhere in genome
(not targeted)
• copy number can be very high
• transgenic animals are usually random
integrations

17. Homologous
Recombination:
• driven by the DNA sequences (targeted)
• rare (1000X less frequent than random)
• increased efficiency
• results in:
- endogenous DNA replaced with exogenous
- specific - targeted
- copy number = 1

18. Targeting Constructfor
Positive/Negative Selection
• To make targeting construct:
- a positive selectable marker flanked by
two “arms” of homologous sequence
- a negative selectable marker
outside one homologous arm

19. Positive selectionmarkers
• To enrich recombination events
• Expression cassettes encoding antibiotic resistance genes

20. NegativeSelectionMarkers
• Used to enrich for homologous recombination events over
random insertions.
• Use of Herpes Simplex Virus (HSV) Thymidine Kinase (TK) gene
coupled with gancyclovir treatment

21. Homologousrecombinant

23. Selection Strategy:
• Positive Selection
– G418
- Neomycin Resistance gene
- confers resistance to G418
• Negative Selection
- Gancyclovir
- Herpes Simplex Virus Thymidine Kinase
(HSV-TK)
- sensitive to gancyclovir
- selects against random integrants

25. Making knockoutmice
 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 germ line
 Chimeric mice each heterozygous for the knockout mutation
are mated to produce homozygous knockout mice

26. Procedure for making mixed
genotype blastocyst

27. Breedingscheme forproducingknockoutmice.

31. Drawbacksof knockoutmice
• 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

32. Conditionalknockoutapproach
• Delete the gene of interest in a time and space- dependant
manner using site-specific recombinases.
• Using these recombinases it is possible to knockout the
expression of a gene in a specific mouse tissue or at a specific
stage of development or in response to an inducer.

33. The Cre-loxP System
• 1987- Brian Sauer’s introduction of the Cre-loxP system for
temporal control of transgenic gene expression
• 1995- K Rajewsky demonstrated “inducible gene targetting in
mice” using the Cre-loxP conditional knockout

34. • Cre recombinase is a site specific integrase isolated from P1
bacteriophage
• Catalyses recombiniation between two of its consensus DNA
recognition sites known as the loxP sites which are 34 bp in
length
• Two 13 bp palindromic sequences that flank a central
sequence of 8 bp which determines the directionality of the
loxP site

37. SpatialandTemporalknockoutisachievedbythe
choiceofpromoterusedtodrivetheCre-gene
expression:
• Several mouse lines each expressing Cre from a promoter that
is either tissue-specific, cell specific, developmentally
specific,or responsive to an exogenous agent like tetracycline
are now available.
• Thus, several promoter specific mouse models can be
generated

40. Knockout vs. Knock in
• In contrast to knockout in which a gene or part of gene is
deleted, knockin is the replacement of the gene by mutant
version of the same gene using homologous recombination.
• Knockin is very useful while establishing a disease model of a
specific disease related mutation in human gene.

41. 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

42.  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

43. 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

44. Knockout mice and cancer
• Rb gene was knocked out but instead of eye tumours the
animals suffered from pituitary gland and thyroid gland
tumours
• It was later found that a second gene protected the eye cells
from cancer and both mutations are required for tumours to
form

45. 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.

47. http://www.dnalc.org/view/16856-Animation-
41-DNA-is-only-the-beginning-for-
understanding-the-human-genome-.html