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Overexpression of RANK in INS1 Cells Using Adenoviral Infection

March 3, 2017
Icahn School of Medicine at Mount Sinai
Sarah DeSouza, Rosemary Li

I. Abstract

This specific experiment will utilize adenoviral infections to monitor the overexpression
of the RANK gene in INS1 cells. The INS-1 cells would be coming from a cell line as opposed
to directly from mice. Adenoviral infections are a group of viruses that can be easily identified as
viruses that cause pinkeye and diarrhea.1 The RANK gene is the receptor for the RANK Ligand
and is a part of the RANK/RANKL/OPG signaling pathway that regulates activation of bone
cells that break down bone tissues.2 The final data collected will be collected through cell
microscopy, which will show areas of the cell and occurring cell death that cannot be viewed by
the naked eye.

II. Background

Largely unknown to the general public, diabetes mellitus affects more people than breast
cancer and AIDs combined. Approximately 1.4 million Americans are diagnosed with diabetes
every year. 176 billion dollars is spent on direct medical costs for patients with diabetes. As of
2010, diabetes was the seventh leading cause of death in Americans. While adults with diabetes
have a fifty percent higher death rate than adults without diabetes, the problem remains largely
unknown by the American population. The problem, at hand, is that diabetes is currently a non
curable disease. While researchers obviously aim to cure the disease, diabetes research is also
focused on perfecting current treatments and monitoring the long-term effects of possible
treatments.
Diabetes originates in the pancreas, which is a gland located in the abdomen, that is a
vital part of the digestive system and also helps control blood sugar levels. The two main
functions of the pancreas are accomplished via cells that serve as endocrine and exocrine glands.

1
2
Wikipedia
The endocrine cells produce and secrete hormones such as insulin and glucagon. The exocrine
glands produce enzymes that help with the digestion of food. Within the pancreas are the islets of
Langerhans, which are composed of only endocrine cells. There are three main types of cells
within the islets of Langerhans: beta cells, alpha cells and delta cells. Beta cells are the only cells
in the body producing the hormone, insulin, which regulates the amount of glucose in the blood.
Because diabetes is the lack of functioning beta cells, beta cells play an integral role in diabetic
research, in general. Diabetes is when you have high amounts of glucose in the blood over an
extended period of time. There are 2 types of diabetes: type I and type II. Type I diabetes is a
lesser expressed disease and occurs when beta cells are producing no insulin. Type II diabetes,
the more common form of diabetes, occurs when insulin is not being produced in sufficient
amounts.

III. Project Description

The objective of this experiment is to explore the effect of the RANK gene on INS1 cells.
In previous research (Kondegowda et al. year), it was found that through the inhibition of the
RANK Ligand pathway (its receptor being RANK), human beta cell proliferation occurred when
osteoprotegerin (protein) and denosumab (drug) also acted as stimulants. Through this
experiment, we seek to observe how the RANK gene works on its own and whether it is possible
that functioning beta cells can be produced without osteoprotegerin and denosumab acting as
catalysts.
The proposed goals will be achieved through a series of methods, including cell culture,
and immunofluorescence cell death assay.

IV. Research Strategy

Day 1 of this experiment will be dedicated to plating INS1 cells in non-infected and viral
infected conditions. There will be four types of conditions for both non-infected and viral
infected. A twenty four well plate will be sterilized and eight coverslips will be placed into plate.
Non-infected conditions will be plated first with 30,000 cells from cell suspension into each well.
The plate will then be left in 37 degrees Celsius.
For the other four viral infected conditions, 120,000 cells will be placed into an
Eppendorf tube. If the volume is greater than 300 microliters, spin down using a centrifuge and
aspirate some media out. Add RANK virus and gently pipette a few times. Incubate at 37 degrees
Celsius for two hours. After two hours, add 1 milliliter of culture media into four wells of the
plate. Pipette infected Eppendorf gently to resuspend the cells then divide equally between the
four walls. Leave overnight at 37 degrees Celsius.
Day 2 will consists of cytokine addition. Cytokines are important to the signaling of cells.
There will be four cytokine-treated conditions which are represented through a cytokine master
mix. This master mix should be added to all the wells and then incubated at 37 degrees Celsius
for another twenty four hours.
Day 3 will be for fixation and staining. Slowly aspirate out cell culture media and PFA
per well. Leave in the dark for 20 minutes. Aspirate PFA and wash with PBS. After waiting a
minute, aspirate out PBS and then wash again with the same amount of PBS. Take out coverslips
for one of the duplicates and block with NGB for one to two hours. Wrap the rest of the plate in
parafilm and place at 4 degrees Celsius.
Day 4 will be dedicated primarily to staining and imaging. Aspirate the primary antibody
and wash with PBS three times for 5 minutes each. Add secondary antibody diluted in NGB and
incubate at real time for 30 minutes. Aspirate and wash with a combination of PBS and Dapi.
Mount the slides and keep in 4 degrees Celsius until you are ready for imaging. The analysis
consists of counting cells that fluoresce because of DAPI staining in comparison those cells that
fluoresce less brightly because of cell death. The percentage will then be calculated.
V. Bibliography

What Is The Pancreas? (n.d.). Retrieved November 12, 2016, from


http://pathology.jhu.edu/pc/BasicOverview1.php?area=ba

Edelman, D. (2013, March 26). Why Do Cancer & AIDS Get More Support Than Diabetes?
Retrieved March 06, 2017, from
https://www.diabetesdaily.com/blog/2013/03/why-do-canceraids-get-more-support-than-diabetes

Bader, E., Migliorini, A., Gegg, M., Moruzzi, N., Gerdes, J., Roscioni, S. S., ... & Aichler, M.
(2016). Identification of proliferative and mature -cells in the islets of Langerhans. Nature,
535(7612), 430-434.

Wang, P., Fiaschi-Taesch, N. M., Vasavada, R. C., Scott, D. K., Garca-Ocaa, A., & Stewart, A.
F. (2015). Diabetes mellitusadvances and challenges in human -cell proliferation. Nature
Reviews Endocrinology,11(4), 201-212. doi:10.1038/nrendo.2015.9

Kondegowda et al., 2015, Cell Metabolism 22, 7785 July 7, 2015 a2015 Elsevier Inc.
http://dx.doi.org/10.1016/j.cmet.2015.05.0

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