Title: Discuss an example of knockout mouse model used for disease modelling Disease: Metastatic Bladder Cancer Module: Gene and Tissue Culture Technology

1. BIO60704
GENES AND TISSUE CULTURE TECHNOLOGY
PRESENTATION TITLE :
DISCUSS AN EXAMPLE OF KNOCK OUT MOUSE
MODEL USED FOR DISEASE MODELLING
Module Lecturer: Dr. Yap Wei Hsum
Student Name:
Mong Jia Ai (0326343)
Sharon Rachel Wong (0326642)
Saniika A/P Renganadan (0326340)
Jesvinder Jeet Kaur (0327298)

2. WHAT IS GENE TARGETING?
• A method for modifying the structure of a
specific gene without removing it from its
natural environment in the chromosome of
a living cell.
• Involves in the construction of a piece of
DNA, known as a gene targeting vector ,
which is then introduced into the cell where
it replaces or modifies the normal
chromosomal gene through the process of
homologous recombination (McQueen,
2018).
• Homologous recombination in embryonic
stem cells to modify the mouse genome at a
specific locus.
• Generating a piece of DNA that is identical to
the locus of interest and this engineered
piece is swapped in to replace the original
piece of DNA.
• Even with this selection, most of the surviving
ES cells have integrated the new piece of DNA
at a random locus rather than recombining it
at the correct locus of interest.
• Due to this, ES cells showing resistance to the
selective agent must also be screened by
Southern blot or by PCR to discover which
clones have been correctly targeted (Koch
Institute for Integrative Cancer Research at
MIT, n.d.).

3. WHAT IS KNOCK IN AND KNOCK OUT MOUSE?
Knock In Mouse
• To study the exogenous expression of a
protein.
• A knock in mouse is generated by targeted
insertion of the transgene at a selected
locus (Koch Institute for Integrative Cancer
Research at MIT, n.d.).
• A gene sequence of interest is altered by
one-for-one substitution with a transgene,
or by adding gene sequences that are not
found within the locus (genOway, n.d.)
Knock Out Mouse
• Researchers have inactivated, or "knocked
out," an existing gene by replacing it or
disrupting it with an artificial piece of DNA.
• The loss of gene activity often causes
changes in a mouse’s phenotype which
includes appearance, behaviour and other
observable physical and biochemical
characteristics (National Human Genome
Research Institute, 2015).

4. RESEARCHERS WHO DEVELOPED THE TECHNOLOGY FOR THE
CREATION OF KNOCKOUT MICE WON THE NOBEL PRIZE IN THE YEAR
2007
• The Nobel Prize in Physiology or Medicine 2007 was awarded jointly to Dr Mario R.
Capecchi, Dr Martin J. Evans and Dr Oliver Smithies “for their discoveries of principles for
introducing specific gene modifications in mice by the use of embryonic stem cells (ES)”.
(1)
Dr Martin Evans
(Cultivation of ES Cells)
Dr Oliver Smithies
(Gene Knockout)
Dr Mario Capecchi
(Gene Targeting)

5. HOW KNOCK OUT MOUSE MODEL IS MADE?
• First strategy: Gene targeting or homologous recombination
(specifically manipulate a gene in the nucleus of an ES cell).
Introducing an artificial piece of DNA that shares identical, or
homologous, sequence to the gene.
This homologous sequence flanks the existing gene's DNA
sequence both upstream and downstream of the gene's
location on the chromosome.
• Second strategy: Gene trapping (a random process is used).
A piece of artificial DNA containing a reporter gene is
designed to insert randomly into any gene.
The inserted piece of artificial DNA prevents the cell's RNA
"splicing" machinery from working properly, thus preventing
the existing gene from producing its designated protein and
knocking out its function.
• Both consists of a modified viral vector or a linear fragment of
bacterial DNA. After the artificial DNA is inserted, the genetically
altered ES cells are grown in a lab dish for several days and
injected into early-stage mouse embryos (National Human
Genome Research Institute, 2015).
Source: Jargon Wall

6. ENHANCED MORTALITY TO METASTATIC BLADDER CANCER CELL LINE
MB49 IN VASOACTIVE INTESTINAL PEPTIDE GENE KNOCKOUT MICE
• To investigate if the metastatic bladder cancer is susceptible to
death with the absence of Vasoactive Intestinal Peptide (VIP) gene
using the knockout mice model (Mirsaidi et al., 2017).
• MB49 murine bladder cancer cell lines where utilized by injecting
into two strains of mice
• A Kaplan–Meier plot was constructed to compare control
C57BL/6 mice and C57BL/6 mice with MB49 vs. VIP KO controls
and VIP KO mice with MB49.
• VIP KO mice had increased mortality compared to C57BL/6 mice
at 4 weeks. The number of ulcers between both groups was not
statistically significant.
• In vitro studies confirms that the presence of VIP in high doses
reduced MB49 cell growth, as well as macrophage inhibitory
factor (MIF), a growth factor in bladder cancer cells. Therefore,
the presence of VIP may accommodate the susceptibility to death
from bladder cancer, and that it manifests its effect through the
down regulation of MIF.
What is VIP?
• A therapeutic agent for bladder cancer, which is a 28-
amino acid peptide that has multiple use of treatment such
as bronchodilatory and anti-inflammatory properties
(Mirsaidi et al., 2017).
• VIP obstructs the proliferation of small-cell lung cancer in
vitro and in vivo in mice and is proven to promote the
nuclear expression of p53 in mouse renal cell carcinomas.
Loss of expression of p53, a tumour suppressor, and its
analogs leads to tumour growth and can also be found in
patients with bladder cancer.
• Using a mouse bladder cancer cell line, MB49, a model is
designed using leg injections of the cancer cells to
investigate if the loss of VIP gene conducts increased
mortality and/or morbidity from bladder cancer
metastases, compared to control C57BL/6 mice.
• An in vitro analysis is conducted to study the effect of VIP
on MB49 cells. It is hypothesized that VIP would diminish
cell growth by decreasing the activity of macrophage
inhibitory factor (MIF), a known growth factor in bladder
cancer cells(Mirsaidi et al., 2017).

7. RESULTS AND DISCUSSION
• Compared to cells grown in the absence of
VIP, those grown in the presence of VIP
also showed decreased extracellular MIF
accumulation (Mirsaidi et al., 2017).
Tumour Measurement
• In Vitro MB49 cells grown in vitro in the
presence of 150 mg/ml VIP illustrated
decreased cell growth when compared to
cells grown in the absence of VIP (Mirsaidi
et al., 2017).
Figure 1. Vasoactive intestinal peptide (VIP) concentration-
dependent changes to bladder cancer cell count in vitro.
The growth in each group was significantly different from control
cells grown in the absence of VIP. p < 0.001 [multiple
comparisons vs. control group (Holm–Šidak method)].
Figure 2. Vasoactive intestinal peptide (VIP) concentration-
dependent changes to MIF in medium. The MIF
concentration in the cells treated with VIP 150 mg was
significantly reduced compared to controls (*p < 0.05).

8. • VIP KO mice have a higher mortality rate
with exposure to MB49 bladder cancer
cell line than C57BL/6 mice ; The
susceptibility to death from bladder
cancer is inhibited by VIP.
• Cell growth decreased, with the
administration of VIP to bladder cancer
cells cultured in vitro, indicating the role
of VIP in cell proliferation through VPAC1
receptor ; a dose dependent decrease in
the proliferation of the bladder cancer cell
line is seen, induced by VIP and the
elaboration of MIF is reduced, which is
known to increase cancer cell proliferation
and angiogenesis.

9. • Kaplan-Meier plot shows statistically
significant (p=0.002) increased
mortality among VIP KO mice injected
with MB49 bladder cancer, while other
mice (VIP KO controls, WT controls and
WT mice injected with cancer) survived
until the end of term; increased
mortality in VIP KO mice is not
associated with an increased number of
pulmonary metastases, weight loss or
tumour size (Mirsaidi et al., 2017).

10. CONCLUSION
• Vasoactive intestinal peptide KO mice have enhanced susceptibility to death from
MB49 bladder cancer.
• Despite the growth and spread of tumours in both VIP KO and C57BL/6 groups
injected with cancer cells, only VIP KO mice died prior to the end of the study.
• Neither VIP KO controls nor WT controls developed or succumbed to cancer.
• The Kaplan– Meier analysis further suggests that the lack of endogenous VIP
production significantly increases the likelihood of death to cancer.
• Both VIP KO and C57BL/6 bladder-injected mice had metastases to the lung;
however, larger and more numerous metastases were seen in C57BL/6 mice.
• Vasoactive intestinal peptide may potentially be a form of treatment of bladder
cancer.

11. Advantages of Knock Out Mouse Model
• Can be used as models for drugs and gene
therapies
• Relatively good models for human disease due to
similarity (85 % genetic similarity to human
genome)
• Very specific endogenous gene has been altered in
such way that it interferes with the normal
expression
• Provides insights into mutations further than
morphological (physiological and behaviourally)
Disadvantages of Knock Out Mouse Model
• Developmental lethality limits studies to embryonic
development – not all genetically altered embryo
can grow into adult mouse
• Unable to characterize last stage
neurodegenerative disorders
• Cost ineffective
• Phenotypic differences from mutations between
the mice and humans (Webprojects.oit.ncsu.edu,
2018).
Ethical Concerns of Knock Out Mouse Model
• Use of animals in science is a controversial issue.
• There are some moral or ethical factors
contributing to genetic manipulation.

12. REFERENCES
• Genetargeting.com. (2018). The Benefits and Drawbacks of the Transgenic Mouse Model. [online] Available at:
https://www.genetargeting.com/transgenic/transgenic-mouse-model/ [Accessed 22 Oct. 2018].
•
genOway. (n.d.). Knockin Mouse Models. [online] Available at: https://www.genoway.com/services/customized-
mouse/knockin-models/overview.htm [Accessed 21 Oct. 2018].
• Koch Institute for Integrative Cancer Research at MIT. (n.d.). Knockins and Knockouts. [online] Available at:
https://ki.mit.edu/sbc/escell/models/knock [Accessed 19 Oct. 2018].
• McQueen, C. (2018). Comprehensive toxicology. 3rd ed. Oxford, UK: Elsevier, p.709.
• Mirsaidi, N., Burns, M., McClain, S., Forsyth, E., Li, J., Dukes, B., Lin, D., Nahvi, R., Giraldo, J., Patton, M., Wang, P., Lin, K., Miller,
E., Ratliff, T., Hamidi, S., Crist, S., Takemaru, K. and Szema, A. (2017). Enhanced Mortality to Metastatic Bladder Cancer Cell
Line MB49 in Vasoactive Intestinal Peptide Gene Knockout Mice. Frontiers in Endocrinology, [online] 8, pp.1-6. Available at:
<https://www.frontiersin.org/articles/10.3389/fendo.2017.00162/full> [Accessed 21 Oct. 2018].
• National Human Genome Research Institute. (2015). Knockout Mice. [online] Available at:
https://www.genome.gov/12514551/knockout-mice-fact-sheet/ [Accessed 19 Oct. 2018].
• Samarasinghe, B. (2013). The Knockout Mouse. [online] Jargon Wall. Available at: https://www.jargonwall.com/molecular-
biology/knockout-mouse/ [Accessed 21 Oct. 2018].
• Webprojects.oit.ncsu.edu. (2018). genome.gov | Knockout Mice. [online] Available at:
http://webprojects.oit.ncsu.edu/project/bio183de/Black/genetech2/genetech2_news/knockouts.html [Accessed 22 Oct.
2018].
• Yang, B. (2018). Three scientists who invented gene knockout technology won Lasker Basic Medical Research Award.
[online] Discoverymedicine.com. Available at: http://www.discoverymedicine.com/Benjamin-Yang/2009/05/10/three-
scientists-who-invented-gene-knockout-technology-won-lasker-basic-medical-research-award/ [Accessed 22 Oct. 2018].