1. 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

2. 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.

3. 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

4. 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/ http://www.synapticspeculations.com/facts-behind-antibiotic-resistance/

5. 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] Data above courtesy of Evrogen. Retrieved from: http://www.evrogen.com/products/TurboFP635/TurboFP635_Detailed_description.shtml

6. 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: NFAT- exClucTM/GFP – SV40-tdr/rsRluc or NF97 for short. We seek to create: NFAT-exClucTM/GFP – SV40-TurboFP635 (NF98) NFAT-exClucTM/GFP – PGK-TurboFP635 (NF99)

7. 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-fluorescent- nih-nigms.html

8. 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

9. Materials Used: Reactants: NF97, TurboGFP, HindIII, ASCI, NdeI, EcoRI, NheI, dNTP, Klenow polymerase. Buffers: NE Buffer 4, NE Buffer 1, BSA, CIP, nuclease free dH 2 O. Image courtesy of Ross Lippert, retrieved from:http://snhs-plin.barry.edu/cell-biology- lab/Restriction_Digest_MIT_Lippert.htm

10. 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

11. 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.rwth- aachen.de/

12. 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 http://www.jlindquist.net/generalmicro/GBimages/eaurescens.jpg

13. 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!

14. brightfield GFP TurboRFP635 293T control293T – NF98(SV40 promoter) 08-24-2013 NF98 cl18 transfection

15. 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.

16. References [1] Ponomarev V. et al. Imaging TCR-Dependent NFAT- Mediated 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. 23 rd 2012. [4] MacLachlan A. How a Jellyfish Protein Transformed Science Live Science Retrieved from: http://www.livescience.com/16752-gfp-protein-fluorescent- nih-nigms.html http://www.livescience.com/16752-gfp-protein-fluorescent- nih-nigms.html