Gtc presentation - Knockout Mouse Model (group 6)
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Transcript of Gtc presentation - Knockout Mouse Model (group 6)
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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
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IntroductionWhat 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.
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● 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
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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
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● 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
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&
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.
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ApplicationsTable 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
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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
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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.
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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.
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● 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.
>> Gene does not adopt identical functions in mouse and humans.
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Current development
Figure 11: Genetic engineering of insulin. Source - Buck Ball, n.d.
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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.
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ReferencesArnold, 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/>
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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>.