Knockout mice are genetically engineered mice that have had one or more of their genes made defective or inactivated through gene targeting. This allows researchers to study the effects of removing or altering the gene. One example is insulin receptor knockout mice, which are used as a model for diabetes since they lack the insulin receptor gene and exhibit hyperglycemia and insulin resistance. Knockout mice have provided insights into gene function and disease mechanisms and are useful for testing potential new drug therapies. However, some limitations include developmental defects in the mice and differences in gene function between mice and humans.

1. Discuss an example of knockout mouse
model used for disease modeling
Loh Wing How (0327398)
Nathasha Kamurzaman (0320290)
Kan Jun Fai (0327059)
Kirubhan Nadarajan (0327849)
Kesshmita (0328469)
SCT60103 - Genes & Tissue Culture Technology

2. Introduction
What is gene knockout?
● The genetically engineered organism has one or more genes in its chromosomes
that been made to be defective.
● It is a functional tool to study the modifications that can be done to a genome of
a living organism by observing the changes when the gene is absent or when the
mutant gene copy is expressed.

3. ● The targeting vector of the gene :
- pieces of DNA that are homologous
- positive and negative selection markers
Neomycin
phosphomycintransferase (neor)
gene
&
HSV thymidine kinase (HSV-
tk) gene
Figure 2: Knockout Mouse (replacement
vector) - Samuel Kwatia, 17 November
2014
Figure 1: Knockout Mouse (positive
and negative selection markers -
Samuel Kwatia, 17 November 2014

4. Knockout Mouse
● A laboratory mouse in which the gene inactivated (knocked
out) by replacing or distorted with artificial DNA.
● It can cause changes in appearances, behavior, physical and
biochemical characteristics.
● Examples of research of knockout mice in disease modelling:
❏ Cancer
❏ Heart disease
❏ Diabetes
Figure 3: Knockout Mice Fact Sheet -
NHGRI, 27 August 2015

5. ● Knockout mice also offer a biological factors in which drugs and other
therapies can be developed and tested.
● Knockout mice are also inexpensive, easy to raise and have a short
generation time.
Figure 4: The Nobel Prize in
Physiology or Medicine -
Nobelprize.org, 2007

6. &
Production of Knockout Mice
Figure 5: Knockout mice of gene targeting- Eli Owens, n.d. Figure 6: Knockout mice of gene trapping - Eli Owens, n.d.

7. Applications
Table 1 : Insulin action in muscles, adipose tissue, liver, and pancreatic β cells and phenotypes of knockout mice - Takashi Kadowaki , 2000.
Animals Insulin action Insulin secretion Phenotype
Muscles Liver Adipose
tissue
IR knockout Defective Defective Defective Hyperinsulinemia Severe
diabetes

8. Conditional gene modification = only in subset of tissue / only at particular time
1.Cre Lox Technology
Figure 7 : CreLoxP experiment - Matthias Zepper, 2008
2. Flippase (Flp) Recombinase
● Similiar to Cre Lox Technology.
● Uses flippase recombinase from
Saccharomyces cerevisiae.
Flp recognizes Flp recombinase target (FRT)
that flank genomic region of interest
- Flippase = Cre
- FRT= LoxP

9. Defects in Muscle, Fat, and Liver Insulin Receptors in Knockout Mice
Figure 8 : Defects
in muscle, fat and
liver insulin
receptors in
knockout mice -
Linda Willis, 2015.

10. Challenges
● Ethical and moral issues
>> Genetic testing on fetus
- Tay-Sachs disease & sickle-cell anemia
>> Impact on the individual's well-being
● Developmentally lethal (~15%)
>> Not all genetically altered embryos cannot
grow into adult mice
Figure 9: Genetic testing on fetus, n.d.
Figure 10 : Red blood cells and sickle cells.

11. ● Serve a different function in adults than in developing embryos
● Fail to produce observable changes in mouse
● Some genes are difficult to knockout
● Developmental defects
● Produce different characteristics in humans
>> p53 knockout mouse model
>> p53 gene has been silenced
>> Mutations of this gene have been implicated in more than half of all human cancers
>> Tumours will develop a different range of tumours.
>> Example the mice will develop lymphomas and sarcomas whereas humans would develop
epithelial-cell derived cancers.

12. Current development
Figure 11: Genetic engineering of insulin. Source - Buck Ball, n.d.

13. Conclusion
● Mouse model is one of the best model of mammalian development as it is very
ideal in studying the functions of human genes in health and disease
perspective.
● 98 % of the mouse genes have comparable genes as such in human genes,
which allows the researchers to test on certain genes by monitoring how the
particular genes able to regulate.
● The mouse model shares the same features as human based on the physiology
and anatomically development
● Mouse model acts as a representation on medical discovery and for
therapeutic development to cure diseases based on novel target of the human
genome.

14. References
Arnold, P. (23 August 2009). The Scientific Problems of Knockout Mice. Bright Hub, viewed 21 October 2017.
<http://www.brighthub.com/science/genetics/articles/46498.aspx>
Ball, B. (2016). DNA Manipulation Diabetes Genetic Engineering – Animals – Drugs Bacteria Plasmid Biopharming
Transgenic Organisms Knockout Mice Cloning, Slideplayer, viewed 19 October 2017.
<http://slideplayer.com/slide/8067093/>
Bruning, J.C., Baudler, S., Krone, W., Plum, L. & Wunderlich, F.T. (11 May 2015).Transgenic and Knockout Mice in
Diabetes Research: Novel Insights into Pathophysiology, Limitations, and Perspectives. Physiology, 20(3), 152-161.
[online], viewed 18 October 2017. <http://physiologyonline.physiology.org/content/20/3/152.long>
Full Circle Health Care n.d., Genetic Testing, viewed 19 October 2017,
<http://www.fullcirclehealthcareinc.com/genetic-testing.html>
Kadowaki, T. (15 August 2000). Insights into insulin resistance and type 2 diabetes from knockout mouse models.
Journal of Clinical Investigation, 106(4), 459-465. [online], viewed 18 October 2017.
<https://www.ncbi.nlm.nih.gov/pmc/articles/PMC380257/>

15. Kwatia, S. (17 November 2014). Gene Knockout, Slideshare, viewed 18 October 2017.
<https://www.slideshare.net/nanayawsam/gene-knockout-41679109>
National Human Genome Research Institute 2015, Knockout Mice, viewed 19 October 2017,
<https://www.genome.gov/12514551/knockout-mice-fact-sheet/#al-3>
Nobelprize, 2007.The Nobel Prize in Physiology or Medicine 2007, viewed 18 October
2017.<https://www.nobelprize.org/nobel_prizes/medicine/laureates/2007/advanced.html>
Owens, E. n.d. Knockout Mice, Venggage, viewed 18 October 2017.
<https://infograph.venngage.com/p/222718/knockout-mice>
Rajan, R.M. (19 April 2015). Gene knockout animal models, Slideshare, viewed 18 October 2017.
<https://www.slideshare.net/RinuMaryRajan/gene-knockout-animal-models>
Willis, L. (2015). Knockout Mice as a Tool to the Understanding of Diabetes Mellitus, Slideplayer, viewed 19
October 2017. <http://slideplayer.com/slide/7103145/>
Zepper, M. (30 January 2008). CreLoxP experiment, Wikimedia Commons, viewed 21 October 2017.
<https://commons.wikimedia.org/wiki/File:CreLoxP_experiment.png>.