Comparative Analysis of Retroviral Promoters for Reporter gene Imaging of T cell Immunotherapy

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Comparative Analysis of Retroviral Promoters for
Reporter gene Imagingof T cell Immunotherapy

• By Dario Pinos, Jason Lee PhD

Image courtesy of Live Science. Retrieved from
http//www.livescience.com/16752-gfp-protein-fluor
escent-nih-nigms.html
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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.

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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.co
m/case-studies
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Introduction (contd)

• 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.co
m/facts-behind-antibiotic-resistance/
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Introduction (contd)

• 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/TurboFP
635_Detailed_description.shtml
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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. Ponomarevs 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)

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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
fromhttp//www.livescience.com/16752-gfp-protein-
fluorescent-nih-nigms.html
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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
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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
fromhttp//snhs-plin.barry.edu/cell-biology-lab/R
estriction_Digest_MIT_Lippert.htm
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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
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Procedure (contd)

• 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
/
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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/generalm
icro/GBimages/eaurescens.jpg
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Preliminary Results (contd)

• 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!

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

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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-Inducib
  le 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 Live Science Retrieved from
  http//www.livescience.com/16752-gfp-protein-fluor
  escent-nih-nigms.html