Knockout mice are genetically engineered mice that have had specific genes inactivated through gene targeting. This document provides an overview of knockout mice, including their history, generation process, and uses. It describes how embryonic stem cells are isolated and genetically modified through homologous recombination before being injected into blastocysts to generate chimeric mice. Knockout mice are valuable research tools for studying gene function and modeling human diseases. They have contributed significantly to our understanding of immunology and the development of humanized antibody therapies.

1. WELCOME

2. KNOCKOUT MICE
TARA SINGH RAWAT

3. INTRODUCTION
• A knockout mice 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
technique that utilizes homologous recombination to modify the
genome of a living organism.
• Knockouts are basically used to study the function of specific genes.

4. CONT’D….
• Other forms of gene disruption -
• gene knockdown - reduced expression of the gene
• knock-in - replace one allele with another
• Simultaneously knocking out
• 2 genes → double knock out
• 3 genes → triple knockout
• 4 genes → quadruple knockout

5. HISTORY
1989 - Birth of the knockout mouse – the beginning of a new era in biotechnology

6. HISTORY
• 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 Sir Martin J. Evans Oliver Smithies
Gene Knockout Cultivation of ES Cells Gene Targeting

7. GENERATION OF KNOCKOUT
MOUSE
ES cell isolation and culture
Construction of targeting vector
ES cell transfection
Proliferation of targeted ES cells
Injection of transformed cells into blastocysts
Birth and breeding of mosaic mice

8. PRESS RELEASE 2007, The Nobel Assembly at Korolinska Institute
•

9. PRESS RELEASE 2007, The Nobel Assembly at Korolinska Institute
•

10. EMBRYONIC STEM CELL ISOLATION

11. EMBRYONIC STEM CELL CULTURE
• Embryonic stem (ES) cells are isolated from the inner cell mass of a blastocyst.
• ES cells in culture remain undifferentiated provided that they are grown well
separated from each other.
• The presence of the cytokine leukaemia inhibitory factor (LIF) is essential to
ensure that ES cells do not differentiate in vitro.
• For this reason, ES cells are generally grown on a feeder layer of fibroblasts
which secrete LIF into the culture medium.
• Most ES cells lines currently in use have been derived from the 129 strain of
mouse which has an agouti coat colour genotype ; this is useful when identifying
chimeric mice

12. ES CELL COLONIES GROWING ON A LAYER OF FIBROBLAST
FEEDER CELLS. HEALTHY, UNDIFFERENTIATED ES CELLS

13. GENE KNOCKOUT TECHNOLOGY
• Gene targeting - Homologous Recombination
Site specific recombination
Cre/LoxP system – conditional knockout
• Gene trapping - Random integration
• Advanced Technology
CRISPR/CAS9 TECHNOLOGY
TALEN
Zinc Finger Nucleases

14. TARGETING CONSTRUCT FOR
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
• Positive selection markers
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 Thymidine Kinase (HSV-TK) gene coupled with
gancyclovir treatment

16. HOMOLOGOUS RECOMBINAYION

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

18. MAKING MIXED GENOTYPE
BLASTOCYST

19. INJECTION OF TARGETED ES CELLS
INTO BLASTOCYSTS
The blastocyst is held on the holding pipette by gentle suction (1).The injection needle containing
ES cells is advanced into the blastocyst cavity (blastocoel) (2).where the ES cells are released (3)
and the injection needle is removed (4). (John M Walker and Ralph, 2009)

20. 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, and the newly engineered ones.
• These blastocysts are then implanted into the uterus of female mice, to
complete the pregnancy.
• The new born mice will therefore be chimeras: parts of their bodies
result from the original stem cells, other parts result from the engineered
stem cells.

21. BREEDING METHOD FOR
OBTAINING HOMOZYGOUS
KNOCKOUT
Knockout
mouse
http://www.bio.davidson.edu/genomics/method

22. DRAWBACKS OF CONVENTIONAL
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 lead to the failure
of establishment of knockout mouse strain for analysis

23. CONDITIONAL KNOCKOUT APPROACH
– 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 targeting in mice” using
the Cre-loxP conditional knockout
• 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.

24. • Spatial and Temporal knockout is achieved by the choice of promoter used to
drive the Cre-gene expression-
• mouse lines are 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
(Daniel Metzger et al., 2001)

25. What are knockout mice used for-
Nearly every human gene has a counterpart in the mouse. This made the mouse the major model for
elucidating the ways in which our genetic material encodes information.
• Discovery of gene function- functional genomics
• Studying and modeling different kinds of cancer, obesity, heart disease, diabetes, arthritis, substance
abuse, anxiety, aging and Parkinson disease.
• Knockout mice also offer a biological context in which drugs and other therapies can be developed and
tested.
• Development of mouse models-
• The p53 knockout mouse is named after the p53 gene, which codes for a protein that normally suppresses
the growth of tumors by arresting cell division.
• Humans born with mutations that inactivate the p53 gene suffer from Li-Fraumeni syndrome, a
condition that dramatically increases the risk of developing bone cancers, breast cancer and blood cancers
at an early age.
• Other mouse models are named, often with creative flair-
Methuselah - longevity
Frantic - anxiety disorders.

26. IMMUNOLOGICAL IMPORTANCE
• The study of human immunobiology in vivo is limited
• Animal models with humanized immune system has significantly advanced
our understanding on human immunobiology and immune-related diseases
such as autoimmune diseases, virus infections, as well as tumour and graft
rejections.
• Immunodeficient mice with constituted human immunity have been
developed to overcome these constraints
• Now a days every human disease is studied in Mice.
• Also used to produce human antibody, humanized antibody, many other type
of human-mice chimeric antibodies.
(Baojun Zhang et al., 2009)

27. PRODUCTION OF HUMAN ANTIBODY IN
KNOCKOUT MICE

28. THE CHARACTERISTICS OF GENETICALLY
MODIFIED SCID OR RAGNULL MICE
Baojun Zhang et al., 2009

29. THE POTENTIAL STRATEGIES TO
IMPROVE HUMANIZED MOUSE MODEL
Baojun Zhang et al., 2009

30. IgG1 PROTECTS AGAINST RENAL DISEASE IN A MOUSE
MODEL OF CRYOGLOBULINAEMIA
Richard T. Strait et al., 2015, Nature.
Observations-
• Immunization of wild-type mice with a potent immunogen, goat anti-mouse IgD
antiserum (GaMD), leads to a large, rapid, predominantly IgG1 antibody response to goat
IgG (GIgG) and the generation of mouse IgG1–GIgG immune complexes, but no
noticeable disease.
• In contrast, IgG1-deficient mice develop renal disease characterized by increased urinary
protein, leukocyte esterase and erythrocytes (blood), as well as increased blood
concentration of urea (BUN) and decreased serum albumin, with anasarca (subcutaneous
oedema) and peritoneal effusion (Fig. a–e and Extended Data Fig. a).
• Kidney colour in these mice changes from red/brown to yellow, reflecting dramatically
decreased perfusion. (Fig.f).

31. GAMD-IMMUNIZED IgG1 KNOCKOUT MICE DEVELOP LETHAL
GLOMERULOPATHY.

32. GLOMERULOPATHY IN GAMD-IMMUNIZED IgG1– MICE IS COMPLEMENT AND
FCR-Γ INDEPENDENT AND ASSOCIATED WITH IgG3 CRYOGLOBULINAEMIA.
• Lack of the normally dominant IgG-1 response in GaMD-immunized mice was
accompanied by increased production of IgG3, IgM and, IgG2a.
Only cryoprecipitates from IgG1 knockout mice contained detectable IgG3 (brown) in glomerular
capillaries (arrows)

33. CONCLUSION
• These finding shows that immune complexes can destroy kidney function by precipitating in
glomerular capillaries
• Thus, mouse IgG1 probably suppresses disease mediated by complement and FcγRs, as well as
disease mediated by excessive intravascular formation of insoluble immune complexes.
• Ig isotypes that poorly activate effector mechanisms protect against disease caused by more
proinflammatory isotypes.
• Functional similarities between mouse IgG1 and human IgG4 suggest that these observations in
mice are applicable to humans.
• These considerations raise the possibility of using human IgG4 antibodies to suppress
autoimmune and immune complex disorders that are mediated by other isotypes.
• Now this clear that these all finding are only possible by use of Konckout mice technology.

34. REFERENCES
• KAPOOR AND MADHU DIKSHIT, 2005, Transgenic and Gene-Knockout Rodents as
Research tools for Cardiovascular Disorders, Scand. J. Lab. Anim. Sci., Vol. 32.
• BRIAN, R. S., Biotechnology, 2006, Greenwood Press Westport, Connecticut London,
pp138, Science 101, ISSN 1931–3950.
• BAOJUN ZHANG, ZIYUAN DUAN AND YONG ZHAO, 2009, Mouse models with
human immunity and their application in biomedical research , J. Cell. Mol. Med. Vol
13, No 6, pp. 1043-1058
• RICHARD T. STRAIT et al., 2015, IgG1 protects against renal disease in a mouse
model of cryoglobulinaemia, Nature, vol 517.