1. Molecular Imaging Alfred Song National Science Foundation Integrative Graduate Education Research Traineeship Program The University of Texas at Austin The University of Texas MD Anderson Cancer Center

2. Gelovani’s Group Juri Gelovani –Department of Experimental Diagnostic Imaging –Among the first to develop a PET reporter gene for in vivo molecular imaging –Previously at Memorial Sloan-Kettering –Current: In vivo imaging, drug development, molecular biology Research Group –~30 people –Medicine, chemistry, biology, pharmacology, computer science www.mdanderson.org

3. Stains Are Useful for Imaging Biological Tissues Used because tissues, unless pigmented, are low in contrast. Dyes and vital stains are colored or fluorescent chemicals or antibodies that bind selectively to certain molecules and increase contrast. Vital stains keep the cell alive, most others kill the cell. They are rare. www.neurostructural.org/services.htm retina.umh.es/Webvision/VisualCortex.html

4. Reporter Genes: Another Way to Increase Contrast The reporter gene produces a protein product which is able to signal where, when, and in what quantity it is being translated. Reporter genes are inherently vital i.e. keeps your cells alive Reporter genes allow to image structure and function. Attach a reporter to your gene of interest. –Fusion Gene is your gene of interest fused to your reporter gene –Fusion Protein is the expression of your Fusion Gene

5. Fusion Genes Gene or Regulatory Region of InterestReporter Fusion Gene life.nthu.edu.tw/~labwwc/staff.science.uva.nl/~zoon/sms/pictures/gfp.jpg Fusion Protein Transcription Translation www.mshri.on.ca/nagy/gallery.htm Reporter (GFP) Mouse Electroporation, Recombination, Selection, Mating, $30,000.

6. GFP Driven by the Engrailed promoter genetik.fu-berlin.de/institut/en_GFP_fly3.jpg

7. Commercially Available GFP Fish www.beverlytang.com/photos/gfp_fish.jpg

8. Alba the GFP Bunny bioephemera.com/wp-content/uploads/2007/04/albagreen.jpg

9. Aequorea victoria www.conncoll.edu/ccacad/zimmer/GFP-ww/aequorea.jpg Shimomura O, Johnson F, Saiga Y (1962). "Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea". J Cell Comp Physiol 59: 223-39

10. Fluorescence Reporters Come in Many Colors Dunn 2006

11. Other Reporter Genes Bioluminescence reporter genes –Enzymes from fireflies catalyze a reaction that produces light. PET reporter genes –Does not emit light, but traps radiopharmaceuticals inside cells that possess the reporter gene. –This reporter is also an enzyme. It changes the polarity of the radiopharmaceutical to trap it inside the cell.

12. Bioluminescence Reporter Gene Enzyme: Firefly Luciferase Substrate: Luciferine Luciferase catalyzes the reaction of luciferin and ATP into Adenyl-luciferin. When this molecule degrades it produces light. www.britannica.com www.sigmaaldrich.com/img/assets/4201/Luciferase_Reaction_Scheme.gif

13. Transgenic Luciferase Tobacco Plant www.answers.com/topic/glowing-tobacco-plant-jpg In vivo imaging is possible. Water the plant with a solution containing luciferin and it begins to glow.

14. Luciferase Complimentation Imaging (LCI) A method to image molecular interactions. Break luciferase into two pieces, make two fusion genes. If fusion genes interact, luciferase pieces will interaction and signal. Luker et al., 2004

15. Luciferase Complimentation Imaging (LCI) Feed the cells or organism luciferin If the cell lights up, you know that the two proteins interact under the specified conditions If the cell does not light up, your conditions may not cause interaction

16. Positron Emissions Tomography (PET) Reporter Gene Enzyme: Herpes simplex virus thymidine kinase (HSV-TK) is an enzyme that adds phosphates to thymidine. Substrate: Radioactive thymidine analogues (radiopharmaceuticals) are trapped within the cell when phosphorylated. Radioactivity is imaged in vivo via clinical or microPET.

18. Three Dimensional Cell Culture Classically cell culture has been on flat plates. 3D culture mimics the three dimensional environment in tissues of the body. 3D cell culture yields differing results for stress experiments than 2D cultures. –Cells are more resilient to insult –Possibly due to HSP expression in a hypoxic cores and/or cell-cell signaling.

19. 3D Culture: Spheroids Gelovani et al., 2007

22. Gelovani has a stream-lined spheroid culture technique, as well as an arsenal of reporter genes, and a full time, experienced molecular biologist (Najjar). Plans are in place to begin co-culturing cells with an endothelial cell line which they hope will produce a vascular network. 3D Culture: Spheroids

23. Multiphoton Microscopy (MPM) A technique that could combine enhanced fluorescence imaging, ablation by laser, and possibly in vivo optical imaging. Characteristics of MPM –Better depth penetration –Simultaneous imaging of multiple fluorescence reporter genes –Cellular and subcellular laser surgery/ablation Currently, Gelovani is not able to image through the other side of the spheroid

24. Multiphoton Microscopy www.aecom.yu.eduwww.childrensmrc.org/images/upload/multi1(1).JPG photonics.light.utoronto.ca/img_fac/image025.jpg Lasers: Ti:Sapphire Pulsed N 2 ~$150K 100 fs pulse duration

25. Multiphoton Microscopy www.loci.wisc.edu/multiphoton/mp.html Combination 2-photon (red and green) and 3-photon (blue) image of C.elegans embryo Centonze,V.E and J.G.White. (1998) Biophysical J. 75:2015-2024 Images of acid fucsin stained monkey kidney taken at a depth of 60 µm by confocal (left) and multiphoton microscopy (right).

27. Benefits of Collaboration A combination Necrosis/Apoptosis reporter gene In vitro 3D cell culture and in vivo experiments to investigate the relative role of necrosis and apoptosis to thermal insult

28. Conditions for Collaboration Development of Necrosis/Apoptosis reporter gene Construction or access to MPM –Cost: ~$200,000 and a semester to build –Adela Ben-Yaker (ME dept) is an expert on ultrafast lasers and has done MPM ablation in the past –IGERT facilities have an MPM, but charges a fee