Illustrating Molecular Events with Light: A Perspective on Optical Reporter Genes

  • Pritha Ray


A major medical success achieved in the last century was the decline of the overall mortality rate of cancer patients by providing high-quality medical care. Many groundbreaking discoveries along with imaging formed the base of this achievement in modern medicine. Noninvasive molecular imaging using small animals is a relatively recent field as compared to clinical imaging in biomedical science. Rapid development in imaging strategies and imaging instrumentations quickly brought this field to the forefront of modern medicine and is now contributing to the development of the very basic steps of clinical imaging. Noninvasive molecular imaging can be categorized into direct and indirect imaging. Theoretically, indirect imaging involves reporter genes that after introduction in cells or animals indirectly measure the expression of an endogenous gene or promoter, while direct imaging engages those molecular probes that attempt to monitor a specific molecular process (e.g., receptor/ligand binding) in living cells or animals.


Reporter Gene Fluorescence Resonance Energy Transfer Bioluminescence Imaging Cell Trafficking Fluorescence Imaging System 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



The author sincerely acknowledges the mentorship, guidance, and encouragement from Prof. Sanjiv Sam Gambhir throughout her research in this exciting field of molecular imaging. The author further wishes to express her gratitude to Drs. Abhijit De and Drs. Shariar Yaghoubi for their help and for the critical review of the manuscript and thanks all of her colleagues at Stanford and UCLA for their help and support.


  1. Adams, J. Y., M. Johnson, M. Sato, F. Berger, S. S. Gambhir, M. Carey, M. L. Iruela-Arispe, and L. Wu. 2002. Visualization of advanced human prostate cancer lesions in living mice by a targeted gene transfer vector and optical imaging. Nat Med 8 (8):891–7.PubMedGoogle Scholar
  2. Afanasieva, T. A., M. Wittmer, A. Vitaliti, M. Ajmo, D. Neri, and R. Klemenz. 2003. Single-chain antibody and its derivatives directed against vascular endothelial growth factor: application for antiangiogenic gene therapy. Gene Ther 10 (21):1850–9.CrossRefPubMedGoogle Scholar
  3. Bhaumik, S., and S. S. Gambhir. 2002. Optical imaging of renilla luciferase reporter gene expression in living mice. Proc Natl Acad Sci U S A 99 (1):377–82.CrossRefPubMedGoogle Scholar
  4. Bradbury, M. S., G. Panagiotakos, B. K. Chan, M. Tomishima, P. Zanzonico, J. Vider, V. Ponomarev, L. Studer, and V. Tabar. 2007. Optical bioluminescence imaging of human ES cell progeny in the rodent CNS. J Neurochem 102 (6):2029–39.CrossRefPubMedGoogle Scholar
  5. Branchini, B. R., M. H. Murtiashaw, T. L. Southworth, S. R. Wilkinson, N. F. KHattak, J. C. Rosenberg, and M. Zimmer. 2005. Mutagenesis evidence that the partial reactions of firefly bioluminescence are catalyzed by different conformations of the luciferase C-terminal domain. Biochemistry 44:1385–93.CrossRefPubMedGoogle Scholar
  6. Brown, E. B., R. B. Campbell, Y. Tsuzuki, L. Xu, P. Carmeliet, D. Fukumura, and R. K. Jain. 2001. In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy. Nat Med 7 (7):864–8.CrossRefPubMedGoogle Scholar
  7. Campbell, R. E., A. E. Palmer, O. Tour, P. A. Steinbach, G. S. Baird, D. A. Zacharias, and R. Y. Tsien. 2002. A monomeric red fluorescent protein. Proc Natl Acad Sci U S A 99:7877–82.CrossRefPubMedGoogle Scholar
  8. Cao, F., M. Drukker, S. Lin, A. Y. Sheikh, X. Xie, Z. Li, A. J. Connolly, I. L. Weissman, and J. C. Wu. 2007. Molecular imaging of embryonic stem cell misbehavior and suicide gene ablation. Cloning Stem Cells 9 (1):107–17.CrossRefPubMedGoogle Scholar
  9. Cao, F., S. Lin, X. Xie, P. Ray, M. Patel, X. Zhang, M. Drukker, S. J. Dylla, A. J. Connolly, X. Chen, I. L. Weissman, S. S. Gambhir, and J. C. Wu. 2006. In vivo visualization of embryonic stem cell survival, proliferation, and migration after cardiac delivery. Circulation 113 (7):1005–14.CrossRefPubMedGoogle Scholar
  10. Chang, S. C., H. L. Chuang, Y. R. Chen, J. K. Chen, H. Y. Chung, Y. L. Lu, H. Y. Lin, C. L. Tai, and J. Lou. 2003. Ex vivo gene therapy in autologous bone marrow stromal stem cells for tissue-engineered maxillofacial bone regeneration. Gene Ther 10 (24):2013–9.CrossRefPubMedGoogle Scholar
  11. Contag, P. R., I. N. Olomu, D. K. Stevenson, and C. H. Contag. 1998. Bioluminescent indicators in living mammals. Nat Med 4 (2):245–7.CrossRefPubMedGoogle Scholar
  12. Day, J. C., L. C. Tisi, and M. J. Bailey. 2004. Evolution of beetle bioluminescence: the origin of beetle luciferin. Luminescence 19 (1):8–20.CrossRefPubMedGoogle Scholar
  13. De, A., and S. S. Gambhir. 2003. PET in imaging gene expression and therapy. Positron Emission Tomography: Basic Science and Clinical practice, Springer Publication, New York, 845–68.Google Scholar
  14. De, A., and S. S. Gambhir. 2005. Noninvasive imaging of protein–protein interactions from live cells and living subjects using bioluminescence resonance energy transfer. Faseb J 19 (14):2017–9.PubMedGoogle Scholar
  15. De, A., X. Z. Lewis, and S. S. Gambhir. 2003. Noninvasive imaging of lentiviral-mediated reporter gene expression in living mice. Mol Ther 7 (5 Pt 1):681–91.CrossRefPubMedGoogle Scholar
  16. De, A., A. M. Loening, and S. S. Gambhir. 2007. An improved bioluminescence resonance energy transfer strategy for imaging intracellular events in single cells and living subjects. Cancer Res 67 (15):7175–83.CrossRefPubMedGoogle Scholar
  17. de Wet, J. R., K. V. Wood, M. DeLuca, D. R. Helinski, and S. Subramani. 1987. Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol 7 (2):725–37.PubMedGoogle Scholar
  18. de Wet, J. R., K. V. Wood, D. R. Helinski, and M. DeLuca. 1985. Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coli. Proc Natl Acad Sci U S A 82 (23):7870–3.CrossRefPubMedGoogle Scholar
  19. Doyle, T. C., S. M. Burns, and C. H. Contag. 2004. In vivo bioluminescence imaging for integrated studies of infection. Cell Microbiol 6 (4):303–17.CrossRefPubMedGoogle Scholar
  20. Edinger, M., Y.A. Cao, M.R. Verneris, M.H. Bachmann, C.H. Contag, and R.S. Negrin. 2003. Blood 101 (2): 640–8.Google Scholar
  21. Edinger, M., T. J. Sweeney, A. A. Tucker, A. B. Olomu, R. S. Negrin, and C. H. Contag. 1999. Noninvasive assessment of tumor cell proliferation in animal models. Neoplasia 1 (4):303–10.CrossRefPubMedGoogle Scholar
  22. Gafni, Y., G. Pelled, Y. Zilberman, G. Turgeman, F. Apparailly, H. Yotvat, E. Galun, Z. Gazit, C. Jorgensen, and D. Gazit. 2004. Gene therapy platform for bone regeneration using an exogenously regulated, AAV-2-based gene expression system. Mol Ther 9 (4):587–95.CrossRefPubMedGoogle Scholar
  23. Galarneau, A., M. Primeau, L. E. Trudeau, and S. W. Michnick. 2002. Beta-lactamase protein fragment complementation assays as in vivo and in vitro sensors of protein protein interactions. Nat Biotechnol 20 (6):619–22.CrossRefPubMedGoogle Scholar
  24. Galperin, E., V. V. Verkhusha, and A. Sorkin. 2004. Three-chromophore FRET microscopy to analyze multiprotein interactions in living cells. Nat Methods 1 (3):209–17.CrossRefPubMedGoogle Scholar
  25. Gilad, A. A., P. T. Winnard, Jr., P. C. van Zijl, and J. W. Bulte. 2007. Developing MR reporter genes: promises and pitfalls. NMR Biomed 20 (3):275–90.CrossRefPubMedGoogle Scholar
  26. Graham, F. L., J. Smiley, W. C. Russell, and R. Nairn. 1977. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol 36 (1):59–74.CrossRefPubMedGoogle Scholar
  27. Greer, L. F., 3rd, and A. A. Szalay. 2002. Imaging of light emission from the expression of luciferases in living cells and organisms: a review. Luminescence 17 (1):43–74.CrossRefPubMedGoogle Scholar
  28. Hastings, J. W. 1983. Biological diversity, chemical mechanisms, and the evolutionary origins of bioluminescent systems. J Mol Evol 19 (5):309–21.CrossRefPubMedGoogle Scholar
  29. Hoffman, R. M. 2005. Advantages of multi-color fluorescent proteins for whole-body and in vivo cellular imaging. J Biomed Opt 10 (4):41202.CrossRefPubMedGoogle Scholar
  30. Hoffman, R. M., and M. Yang. 2006. Whole-body imaging with fluorescent proteins. Nat Protoc 1 (3):1429–38.CrossRefPubMedGoogle Scholar
  31. Honigman, A., E. Zeira, P. Ohana, R. Abramovitz, E. Tavor, I. Bar, Y. Zilberman, R. Rabinovsky, D. Gazit, A. Joseph, A. Panet, E. Shai, A. Palmon, M. Laster, and E. Galun. 2001. Imaging transgene expression in live animals. Mol Ther 4 (3):239–49.CrossRefPubMedGoogle Scholar
  32. Iyer, M., M. Sato, M. Johnson, S. S. Gambhir, and L. Wu. 2005. Applications of molecular imaging in cancer gene therapy. Curr Gene Ther 5 (6):607–18.CrossRefPubMedGoogle Scholar
  33. Iyer, M., L. Wu, M. Carey, Y. Wang, A. Smallwood, and S. S. Gambhir. 2001. Two-step transcriptional amplification as a method for imaging reporter gene expression using weak promoters. Proc Natl Acad Sci U S A 98 (25):14595–600.CrossRefPubMedGoogle Scholar
  34. Jounaidi, Y., J. C. Doloff, and D. J. Waxman. 2007. Conditionally replicating adenoviruses for cancer treatment. Curr Cancer Drug Targets 7 (3):285–301.CrossRefPubMedGoogle Scholar
  35. Kim, Y. J., P. Ray, P. Dubey, S. S. Gambhir, and O. N. Witte. 2004. Multimodality imaging of lymphocytic migration using lentiviral-based transduction of a tri-fusion reporter gene. Mol Imaging Biol 6 (5):331–40.CrossRefPubMedGoogle Scholar
  36. Levenson, R. M., and J. R. Mansfield. 2006. Multispectral imaging in biology and medicine: slices of life. Cytometry A 69 (8):748–58.PubMedGoogle Scholar
  37. Loening, A. M., A. M. Wu, and S. S. Gambhir. 2007. Red-shifted renilla reniformis luciferase variants for imaging in living subjects. Nat Methods 4 (8):641–3.CrossRefPubMedGoogle Scholar
  38. Lu, Y., H. Dang, B. Middleton, Z. Zhang, L. Washburn, M. Campbell-Thompson, M. A. Atkinson, S. S. Gambhir, J. Tian, and D. L. Kaufman. 2004. Bioluminescent monitoring of islet graft survival after transplantation. Mol Ther 9 (3):428–35.CrossRefPubMedGoogle Scholar
  39. Luker, G. D., V. Sharma, C. M. Pica, J. L. Dahlheimer, W. Li, J. Ochesky, C. E. Ryan, H. Piwnica-Worms, and D. Piwnica-Worms. 2002. Noninvasive imaging of protein–protein interactions in living animals. Proc Natl Acad Sci U S A 99 (10):6961–6.CrossRefPubMedGoogle Scholar
  40. Luker, G. D., V. Sharma, C. M. Pica, J. L. Prior, W. Li, and D. Piwnica-Worms. 2003. Molecular imaging of protein–protein interactions: controlled expression of p53 and large T-antigen fusion proteins in vivo. Cancer Res 63 (8):1780–8.PubMedGoogle Scholar
  41. Massoud, T. F., and S. S. Gambhir. 2003. Molecular imaging in living subjects: seeing fundamental biological processes in a new light. Genes Develop 17 (5):545–80.CrossRefPubMedGoogle Scholar
  42. Massoud, T. F., and S. S. Gambhir. 2007. Integrating noninvasive molecular imaging into molecular medicine: an evolving paradigm. Trends Mol Med 13 (5):183–91.CrossRefPubMedGoogle Scholar
  43. Matz, M. V., A. F. Fradkov, Y. A. Labas, A. P. Savitsky, A. G. Zaraisky, M. L. Markelov, and S. A. Lukyanov. 1999. Fluorescent proteins from nonbioluminescent Anthozoa species. Nat Biotechnol 17 (10):969–73.CrossRefPubMedGoogle Scholar
  44. Meighen, E. A. 1993. Bacterial bioluminescence: organization, regulation, and application of the lux genes. Faseb J 7 (11):1016–22.PubMedGoogle Scholar
  45. Mocanu, J. D., K. W. Yip, N. M. Alajez, W. Shi, J. H. Li, S. J. Lunt, E. H. Moriyama, B. C. Wilson, M. Milosevic, K. W. Lo, N. van Rooijen, P. Busson, C. Bastianutto, and F. F. Liu. 2007. Imaging the modulation of adenoviral kinetics and biodistribution for cancer gene therapy. Mol Ther 15 (5):921–9.PubMedGoogle Scholar
  46. Montet, X., J. L. Figueiredo, H. Alencar, V. Ntziachristos, U. Mahmood, and R. Weissleder. 2007. Tomographic fluorescence imaging of tumor vascular volume in mice. Radiology 242 (3):751–8.CrossRefPubMedGoogle Scholar
  47. Ntziachristos, V., J. Ripoll, L. V. Wang, and R. Weissleder. 2005. Looking and listening to light: the evolution of whole-body photonic imaging. Nat Biotechnol 23 (3):313–20.CrossRefPubMedGoogle Scholar
  48. Ozawa, T., A. Kaihara, M. Sato, K. Tachihara, and Y. Umezawa. 2001. Split luciferase as an optical probe for detecting protein–protein interactions in mammalian cells based on protein splicing. Anal Chem 73 (11):2516–21.CrossRefPubMedGoogle Scholar
  49. Paulmurugan, R., and S. S. Gambhir. 2003. Monitoring protein–protein interactions using split synthetic renilla luciferase protein-fragment-assisted complementation. Anal Chem 75 (7):1584–9.CrossRefPubMedGoogle Scholar
  50. Paulmurugan, R., Y. Umezawa, and S. S. Gambhir. 2002. Noninvasive imaging of protein–protein interactions in living subjects by using reporter protein complementation and reconstitution strategies. Proc Natl Acad Sci U S A 99 (24):15608–13.CrossRefPubMedGoogle Scholar
  51. Pike, L., J. Petravicz, and S. Wang. 2006. Bioluminescence imaging after HSV amplicon vector delivery into brain. J Gene Med 8 (7):804–13.CrossRefPubMedGoogle Scholar
  52. Piwnica-Worms, D., D. P. Schuster, and J. R. Garbow. 2004. Molecular imaging of host-pathogen interactions in intact small animals. Cell Microbiol 6 (4):319–31.CrossRefPubMedGoogle Scholar
  53. Prasher, D. C., V. K. Eckenrode, W. W. Ward, F. G. Prendergast, and M. J. Cormier. 1992. Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111 (2):229–33.CrossRefPubMedGoogle Scholar
  54. Ray, P., A. De, J. J. Min, R. Y. Tsien, and S. S. Gambhir. 2004. Imaging tri-fusion multimodality reporter gene expression in living subjects. Cancer Res 64 (4):1323–30.CrossRefPubMedGoogle Scholar
  55. Ray, P., H. Pimenta, R. Paulmurugan, F. Berger, M. E. Phelps, M. Iyer, and S. S. Gambhir. 2002. Noninvasive quantitative imaging of protein–protein interactions in living subjects. Proc Natl Acad Sci U S A 99 (5):3105–10.CrossRefPubMedGoogle Scholar
  56. Ray, P., R. Tsien, and S. S. Gambhir. 2007. Construction and validation of improved triple fusion reporter gene vectors for molecular imaging of living subjects. Cancer Res 67 (7):3085–93.CrossRefPubMedGoogle Scholar
  57. Ray, P., A. M. Wu, and S. S. Gambhir. 2003. Optical bioluminescence and positron emission tomography imaging of a novel fusion reporter gene in tumor xenografts of living mice. Cancer Res 63 (6):1160–5.PubMedGoogle Scholar
  58. Rehemtulla, A., D. E. Hall, L. D. Stegman, U. Prasad, G. Chen, M. S. Bhojani, T. L. Chenevert, and B. D. Ross. 2002. Molecular imaging of gene expression and efficacy following adenoviral-mediated brain tumor gene therapy. Mol Imaging 1 (1):43–55.CrossRefPubMedGoogle Scholar
  59. Remington, S. J. 2006. Fluorescent proteins: maturation, photochemistry and photophysics. Curr Opin Struct Biol 16 (6):714–21.CrossRefPubMedGoogle Scholar
  60. Sahai, E., J. Wyckoff, U. Philippar, J. E. Segall, F. Gertler, and J. Condeelis. 2005. Simultaneous imaging of GFP, CFP and collagen in tumors in vivo using multiphoton microscopy. BMC Biotechnol 5:14.CrossRefPubMedGoogle Scholar
  61. Sato, M., M. L. Figueiredo, J. B. Burton, M. Johnson, M. Chen, R. Powell, S. S. Gambhir, M. Carey, and L. Wu. 2008. Configurations of a two-tiered amplified gene expression system in adenoviral vectors designed to improve the specificity of in vivo prostate cancer imaging. Gene Ther 15 (8):583–93.CrossRefPubMedGoogle Scholar
  62. Serganova, I., and R. Blasberg. 2005. Reporter gene imaging: potential impact on therapy. Nucl Med Biol 32 (7):763–80.CrossRefPubMedGoogle Scholar
  63. Serganova, I., V. Ponomarev, and R. Blasberg. 2007. Human reporter genes: potential use in clinical studies. Nucl Med Biol 34 (7):791–807.CrossRefPubMedGoogle Scholar
  64. Shaner, N. C., R. E. Campbell, P. A. Steinbach, B. N. Giepmans, A. E. Palmer, and R. Y. Tsien. 2004. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22 (12):1567–72.CrossRefPubMedGoogle Scholar
  65. Shaner, N. C., P. A. Steinbach, and R. Y. Tsien. 2005. A guide to choosing fluorescent proteins. Nat Methods 2 (12):905–9.CrossRefPubMedGoogle Scholar
  66. Shu, C. J., S. Guo, Y. J. Kim, S. M. Shelly, A. Nijagal, P. Ray, S. S. Gambhir, C. G. Radu, and O. N. Witte. 2005. Visualization of a primary anti-tumor immune response by positron emission tomography. Proc Natl Acad Sci U S A 102 (48):17412–7.CrossRefPubMedGoogle Scholar
  67. Stagljar, I., C. Korostensky, N. Johnsson, and S. Te Heesen. 1998. A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo. Proc Natl Acad Sci U S A 95 (9):5187–92.CrossRefPubMedGoogle Scholar
  68. Tannous, B. A., D. E. Kim, J. L. Fernandez, R. Weissleder, and X. O. Breakefield. 2005. Codon-optimized Gaussia luciferase cDNA for mammalian gene expression in culture and in vivo. Mol Ther 11 (3):435–43.CrossRefPubMedGoogle Scholar
  69. Thorne, S. H., C. H. Contag. 2005. Using in vivo bioluminescence imaging to shed light on cancer biology. Proc IEEE 93 (4):750–62.CrossRefGoogle Scholar
  70. Troy, T., D. Jekic-McMullen, L. Sambucetti, and B. Rice. 2004. Quantitative comparison of the sensitivity of detection of fluorescent and bioluminescent reporters in animal models. Mol Imaging 3 (1):9–23.CrossRefPubMedGoogle Scholar
  71. Vooijs, M., J. Jonkers, S. Lyons, and A. Berns. 2002. Noninvasive imaging of spontaneous retinoblastoma pathway-dependent tumors in mice. Cancer Res 62 (6):1862–7.PubMedGoogle Scholar
  72. Wahlfors, J., S. Loimas, T. Pasanen, and T. Hakkarainen. 2001. Green fluorescent protein (GFP) fusion constructs in gene therapy research. Histochem Cell Biol 115 (1):59–65.PubMedGoogle Scholar
  73. Weissleder, R., and M. J. Pittet. 2008. Imaging in the era of molecular oncology. Nature 452 (7187):580–9.CrossRefPubMedGoogle Scholar
  74. Winnard, P. T., Jr., J. B. Kluth, and V. Raman. 2006. Noninvasive optical tracking of red fluorescent protein-expressing cancer cells in a model of metastatic breast cancer. Neoplasia 8 (10):796–806.CrossRefPubMedGoogle Scholar
  75. Wood, K. V., Y. A. Lam, H. H. Seliger, and W. D. McElroy. 1989. Complementary DNA coding click beetle luciferases can elicit bioluminescence of different colors. Science 244 (4905):700–2.CrossRefPubMedGoogle Scholar
  76. Wu, J. C., M. Inubushi, G. Sundaresan, H. R. Schelbert, and S. S. Gambhir. 2002. Optical imaging of cardiac reporter gene expression in living rats. Circulation 105 (14):1631–4.CrossRefPubMedGoogle Scholar
  77. Yaghoubi, S. S., R. J. Creusot, P. Ray, C. G. Fathman, and S. S. Gambhir. 2007. Multimodality imaging of T-cell hybridoma trafficking in collagen-induced arthritic mice: image-based estimation of the number of cells accumulating in mouse paws. J Biomed Opt 12 (6):064025.CrossRefPubMedGoogle Scholar
  78. Yaghoubi, S. S., M. C. Jensen, N. Satyamurthy, S. Budhiraja, D. Paik, J. Czernin, and S. S. Gambhir. 2009. Non-invasive detection of therapeutic cytolytic T cells with 18F-FHBG PET in a patient with glioma. Nat. Clin. Pract. Oncol. 6 (1):53–8. Nat Clin Pract Oncol.Google Scholar
  79. Yu, Y. A., T. Timiryasova, Q. Zhang, R. Beltz, and A. A. Szalay. 2003. Optical imaging: bacteria, viruses, and mammalian cells encoding light-emitting proteins reveal the locations of primary tumors and metastases in animals. Anal Bioanal Chem 377 (6):964–72.CrossRefPubMedGoogle Scholar
  80. Zender, L., R. Kock, M. Eckhard, B. Frericks, T. Gosling, T. Gebhardt, S. Drobek, M. Galanski, F. Kuhnel, M. Manns, and S. Kubicka. 2002. Gene therapy by intrahepatic and intratumoral trafficking of p53-VP22 induces regression of liver tumors. Gastroenterology 123 (2):608–18.CrossRefPubMedGoogle Scholar
  81. Zhao, H., T. C. Doyle, O. Coquoz, F. Kalish, B. W. Rice, and C. H. Contag. 2005. Emission spectra of bioluminescent reporters and interaction with mammalian tissue determine the sensitivity of detection in vivo. J Biomed Opt 10 (4):41210.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.Advanced Center for Treatment, Research and Education in Cancer (ACTREC)Tata Memorial Center, KhargharNavi MumbaiIndia

Personalised recommendations