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

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.

● 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

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

● Knockout mice also offer 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

&

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.

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

● Similar 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

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

(DIABETES)

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.

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 can

grow into adult mice

Figure 9: Genetic testing on fetus, n.d.

Figure 10 : Red blood cells and sickle cells.

● 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.

Current development

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

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.

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/>

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>.