Comparative Analysis of Retroviral Promoters for Reporter gene Imaging
of T cell Immunotherapy
By: Dario Pinos, Jason Lee PhD
Image courtesy of Live Science. Retrieved from: http://www.livescience.com/16752-gfp-protein-fluorescent-nih-nigms.html
Acknowledgments
CCNY-MSKCC Partnership for Cancer Research
Dr. Karen Hubbard: Principle Investigator
Nadia Noman: Training Director
Leo Spychala: Administrative Assistant
“This research was supported by NIH/NCI U54CA132378.”
Thank you to my mentors, Jason Lee, PhD and Vladimir Ponomarev, MD, Ph.D
(MSKCC), Juan Zurita (MSKCC), Nikita Suzmin (MSKCC) and Sheila Fortunato
(MSKCC) for their guidance and assistance in this project.
Introduction
What methods are there for treating Cancer?
The most prominent and familiar treatments for cancer would be through the
use of radioactive materials and cytotoxic drugs, radiation and chemotherapy,
respectively. Another process that is being more heavily researched would be
Immunotherapy.
Image courtesy of New Hope Medical Center. Retrieved from: http://www.newhopemedicalcenter.com/case-studies
Introduction (cont’d)
Immunotherapy is the treatment or prevention of disease through the
manipulation of the immune system. For our purposes, we seek to use
molecular imaging methods to track T cell activity for enhanced cancer
immunotherapy.
Image courtesy of Synaptic Speculations. Retrieved from:
http://www.synapticspeculations.com/facts-behind-antibiotic-resistance/
Introduction (cont’d)
• Why fluorescence?
Fluorescence is the emitting of light as a result of exposure to a certain
wavelength. The benefit of using fluorescence is that it provides a
noninvasive method of tracking T-cell activity.[1] By being able to attach
“GFP tags” onto cells allows us to understand the folding process of
proteins and other activities. [4]
Fluorescent
Promoter
Excitation
Maximum (nm)
Emission
Minimum (nm)
Color of Emission
EGFP
488
509
Green
TurboFP635
588
635
Red
Data above courtesy of Evrogen. Retrieved from:
http://www.evrogen.com/products/TurboFP635/TurboFP635_Detailed_description.shtml
How would fluorescence work?
In order to track T cell activity through fluorescence, an effective tag would
have to be created through the use of a Vector, which is a DNA molecule
which is able to transport genetic material from one host to another which
can then be replicated.
Dr. Ponomarev’s lab had previously created a vector called: NFATexClucTM/GFP – SV40-tdr/rsRluc or NF97 for short.
We seek to create:
NFAT-exClucTM/GFP – SV40-TurboFP635 (NF98)
NFAT-exClucTM/GFP – PGK-TurboFP635 (NF99)
Promoters in Question
• The promoters to create NF98 and NF99 are SV40 and PGK, respectively.
• According to scientific literature, when it comes to GFP fluorescence, SV40
consistently performs better in resulting fluorescence in mammalian cells.
[2] [3]
Image courtesy of Live Science. Retrieved from:http://www.livescience.com/16752-gfp-protein-fluorescentnih-nigms.html
Figure 1: Above displays the results from Northeast Normal University, a relationship is
shown between promoter used and the flow cytometry measurement of GFP
fluorescence.
Retrieved from: Qin J. et al. Systematic Comparison of Constitutive Promoters and the
Doxycycline-Inducible Promoter. Plos One 5(5). Published on: May 2010
Materials Used:
• Reactants: NF97, TurboGFP, HindIII, ASCI, NdeI, EcoRI, NheI, dNTP, Klenow
polymerase.
• Buffers: NE Buffer 4, NE Buffer 1, BSA, CIP, nuclease free dH2O.
Image courtesy of Ross Lippert, retrieved from:http://snhs-plin.barry.edu/cell-biologylab/Restriction_Digest_MIT_Lippert.htm
Procedure
• Step 1: Cleaving of NF97 at restriction sites with their respective enzyme.
• Step 2: Gel electrophoresis, isolating desired band lengths, gel purification
of samples.
Image courtesy of Elderkin Conservation Genetics Lab. Retrieved from:
http://www.tcnj.edu/~elderkin/equipment.html
Procedure (cont’d)
• Step 3: Ligation of the desired DNA bands, exposure of the ligation to E.
coli, beginning a transformation process.
• Step 4: Purifying DNA plasmid from several colonies of E. coli.
Image courtesy of RWTHAachen University. Retrieved from: http://www.biologie.rwthaachen.de/
Preliminary Results
Unfortunately once extracted from the E.coli cells, the DNA plasmid created
did not have band lengths matching those that were predicted. However, the
bands across 20 samples were fairly uniform, meaning the transformation
process was successful.
Courtesy of the University of Wisconsin. Retrieved from:
http://www.jlindquist.net/generalmicro/GBimages/eaurescens.jpg
Preliminary Results (cont’d)
However, once the procedure was repeated with the addition of another
restriction enzyme to one of the reactions, one of the colonies of 48 displayed
the desired band lengths!
08-24-2013
NF98 cl18 transfection
brightfield
GFP
TurboRFP635
293T control
293T – NF98(SV40 promoter)
Future Development
Once NF98 is successfully tested, NF99 would be a comparatively shorter
process to create.
The next steps would be to ultimately conduct a series of tests such as
through transformation in mammalian cells to ensure proper orientation of
the vector.
References
[1] Ponomarev V. et al. Imaging TCR-Dependent NFATMediated T-Cell Activation with Positron Emission
Tomography In Vivo Neoplasia 3(6) pp.480-488. 2001.
[2] Qin J. et al. Systematic Comparison of Constitutive
Promoters and the Doxycycline-Inducible Promoter. Plos One
5(5). Published on: May 2010
[3] Liu Q. et al. The Activity of SV40 Promoter can be inhibited
by Overexpression of Heme Oxgenase-1 in Tumor Cells Cell
Biochemistry and Biophysics. Published on Oct. 23rd 2012.
[4] MacLachlan A. How a Jellyfish Protein Transformed Science
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