Nanomedicine pp 99-123 | Cite as

Specific Systems for Evaluation

  • Roderick Adrian SlavcevEmail author
  • Chi Hong Sum
  • Jesse St. Jean
  • Haein Huh
  • Nafiseh Nafissi
Part of the Experientia Supplementum book series (EXS, volume 110)


Fluorescent-based visualization techniques have long been used to monitor biological activity. This chapter explores the delivery of reporter genes as a means to assay and track activity in biological systems. Bioluminescence is the production of light due to biochemical processes. By encoding genes for bioluminescence, biological processes can be visualized based on gene expression. This chapter also discusses the primary applications of bioluminescence as seen through bioluminescent imaging techniques, flow cytometry, and PCR-based methods of gene detection. These techniques are described in terms of researching gene expression, cancer therapy, and protein interactions.


Bioluminescence Luciferase Bioluminescence resonance energy transfer (BRET) Fluorescence resonance energy transfer (FRET) Photoproteins Green fluorescent protein Real-time PCR Reporter genes 


  1. Abbady AQ, Twair A, Ali B, Murad H (2017) Characterization of annexin V fusion with the superfolder GFP in liposomes binding and apoptosis detection. Front Physiol.
  2. Alauddin MM, Gelovani JG (2010) Radiolabeled nucleoside analogues for PET imaging of HSV1-tk gene expression. Curr Top Med Chem 10:1617–1632. Scholar
  3. Alberts B, Johnson A, Lewis J et al (2002) Molecular biology of the cell, 4th edn. Garland Science, New York. Scholar
  4. Alwine JC, Kemp DJ, Stark GR (1977) Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc Natl Acad Sci U S A 74:5350–5354. Scholar
  5. Andersen CL, Jensen JL, Ørntoft TF (2004) Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 64:5245–5250. Scholar
  6. Bakayan A, Domingo B, Miyawaki A, Llopis J (2015) Imaging Ca2+ activity in mammalian cells and zebrafish with a novel red-emitting aequorin variant. Pflugers Arch - Eur J Physiol 467(9):2031–2042. Scholar
  7. Benaron DA, Contag PR, Contag CH (1997) Imaging brain structure and function, infection and gene expression in the body using light. Philos Trans R Soc Lond Ser B Biol Sci 352(1354):755–761. Scholar
  8. Bhaumik S, Lewis XZ, Gambhir SS (2004) Optical imaging of Renilla luciferase, synthetic Renilla luciferase, and firefly luciferase reporter gene expression in living mice. J Biomed Opt 9(3):578–586. Scholar
  9. Bonner WA, Hulett HR, Sweet RG, Herzenberg LA (1972) Fluorescence activated cell sorting. Rev Sci Instrum 43:404–409. Scholar
  10. Boute N, Pernet K, Issad T (2001) Monitoring the activation state of the insulin receptor using bioluminescence resonance energy transfer. Mol Pharmacol 60(4):640–645. Scholar
  11. Boute N, Jockers R, Issad T (2002) The use of resonance energy transfer in high-throughput screening: BRET versus FRET. Trends Pharmacol Sci 23:351–354. Scholar
  12. Brazma A, Hingamp P, Quackenbush J et al (2001) Minimum information about a microarray experiment (MIAME)—toward standards for microarray data. Nat Genet 29:365–371. Scholar
  13. Brogan J, Li F, Li W et al (2012) Imaging molecular pathways: reporter genes. Radiat Res 177:508–513. Scholar
  14. Bullok K, Piwnica-Worms D (2005) Synthesis and characterization of a small, membrane-permeant, caspase-activatable far-red fluorescent peptide for imaging apoptosis. J Med Chem 48:5404–5407. Scholar
  15. Bustin SA, Benes V, Nolan T, Pfaffl MW (2005) Quantitative real-time RT-PCR - a perspective. J Mol Endocrinol 34:597–601. Scholar
  16. Bustin SA, Benes V, Garson JA et al (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622. Scholar
  17. Chalfie M, Tu Y, Euskirchen G et al (1994) Green fluorescent protein as a marker for gene expression. Sci 263:802–805. Scholar
  18. Chou CC, Huang YH (2012) Nucleic acid sandwich hybridization assay with quantum dot-induced fluorescence resonance energy transfer for pathogen detection. Sensors (Switzerland) 12:16660–16672. Scholar
  19. Close DM, Xu T, Sayler GS, Ripp S (2011) In vivo bioluminescent imaging (BLI): noninvasive visualization and interrogation of biological processes in living animals. Sensors 11:180–206CrossRefPubMedGoogle Scholar
  20. Condeelis J, Weissleder R (2010) In vivo imaging in cancer. Cold Spring Harb Perspect Biol.
  21. Contag CH, Contag PR, Mullins JI, Spilman SD, Stevenson DK, Benaron DA (1995) Photonic detection of bacterial pathogens in living hosts. Mol Microbiol 18(4):593–603. Scholar
  22. Contag CH, Ross BD (2002) It’s not just about anatomy: in vivo bioluminescence imaging as an eyepiece into biology. J Magn Reson Imag 16:378–387. Scholar
  23. Cronin M, Akin AR, Collins SA, Meganck J, Kim JB, Baban CK et al (2012) High resolution in vivo bioluminescent imaging for the study of bacterial tumour targeting. PLoS One 7(1). Scholar
  24. Davey HM, Kell DB (1996) Flow cytometry and cell sorting of heterogeneous microbial populations: the importance of single-cell analyses. Microbiol Rev 60:641–696. Scholar
  25. Deluca M, McElroy WD (1974) Kinetics of the firefly luciferase catalyzed reactions. Biochemistry 13(5):921–925. Scholar
  26. Deluca M, McElroy WD (1978) [1] purification and properties of firefly luciferase. Methods Enzymol 57:3–15. Scholar
  27. Deroose CM, Reumers V, Gijsbers R et al (2006) Noninvasive monitoring of long-term lentiviral vector-mediated gene expression in rodent brain with bioluminescence imaging. Mol Ther 14:423–431. Scholar
  28. Fraga H (2008) Firefly luminescence: a historical perspective and recent developments. Photochem Photobiol Sci 7:146–158. Scholar
  29. Gilad AA, Ziv K, Mcmahon MT et al (2008) MRI reporter genes. J Nucl Med 49:1905–1908. Scholar
  30. Gilad AA, Van Laarhoven HWM, Mcmahon MT et al (2009) Feasibility of concurrent dual contrast enhancement using CEST contrast agents and superparamagnetic iron oxide particles. Magn Reson Med 61:970–974. Scholar
  31. Givan A (2011) Flow cytometry: an introduction. Methods Mol Biol 699:1–29. Scholar
  32. Gould SJ, Subramani S (1988) Firefly luciferase as a tool in molecular and cell biology. Anal Biochem 175:5–13. Scholar
  33. Hasegawa S, Furukawa T, Saga T (2010) Molecular MR imaging of cancer gene therapy: ferritin transgene reporter takes the stage. Magn Reson Med Sci 9:37–47. Scholar
  34. Herzenberg LA, Sweet RG, Herzenberg LA (1976) Fluorescence-activated cell sorting. Sci Am 234:108–117. Scholar
  35. Herzenberg LA, Tung J, Moore WA et al (2006) Interpreting flow cytometry data: a guide for the perplexed. Nat Immunol 7:681–685. Scholar
  36. Higuchi-Sanabria R, Garcia EJ, Tomoiaga D et al (2016) Characterization of fluorescent proteins for three- and four-color live-cell imaging in S. cerevisiae. PLoS One. Scholar
  37. Himmelreich U, Dresselaers T (2009) Cell labeling and tracking for experimental models using magnetic resonance imaging. Methods 48:112–124. Scholar
  38. Hofmann A (2010) Spectroscopic techniques: I. Spectrophotometric techniques. In: Principles and techniques of biochemistry and molecular biology. Cambridge University Press, Cambridge, pp 477–521CrossRefGoogle Scholar
  39. Holland PM, Abramson RD, Watson R, Gelfand DH (1991) Detection of specific polymerase chain reaction product by utilizing the 5’----3’ exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci U S A 88:7276–7280CrossRefPubMedPubMedCentralGoogle Scholar
  40. Honigman A, Zeira E, Ohana P, Abramovitz R, Tavor E, Bar I et al (2001) Imaging transgene expression in live animals. Mol Ther 4(3):239–249. Scholar
  41. Jacobs A, Tjuvajev JG, Dubrovin M, et al (2001) Positron emission tomography-based imaging of transgene expression mediated by replication-conditional, oncolytic herpes simplex virus type 1 mutant vectors in vivo. Cancer Res 61:2983–2995. PubMed PMID: 11306477Google Scholar
  42. Jahan-Tigh RR, Ryan C, Obermoser G, Schwarzenberger K (2012) Flow cytometry. J Invest Dermatol 132:1–6. Scholar
  43. Kain SR, Adams M, Kondepudi A et al (1995) Green fluorescent protein as a reporter of gene expression and protein localization. Biotechniques 19:650–655PubMedGoogle Scholar
  44. Kang JH, Chung J-K (2008) Molecular-genetic imaging based on reporter gene expression. J Nucl Med 49:164S–179SCrossRefPubMedGoogle Scholar
  45. Keith M, Farrell P, Behie I, Behie L (2000) Use of flow cytometry to rapidly optimize the transfection of animal cells. Biotechniques 28:14–54CrossRefGoogle Scholar
  46. Lakowicz JR (2006) Fluorophores. In: Principles of fluoescence spectroscopy, 3rd edn. Springer, Berlin, pp 63–69CrossRefGoogle Scholar
  47. Li X, Zhao X, Fang Y et al (1998) Generation of destabilized green fluorescent protein as a transcription reporter. J Biol Chem 273:34970–34975. Scholar
  48. Liu G, Swierczewska M, Niu G et al (2011) Molecular imaging of cell-based cancer immunotherapy. Mol Biosyst 7:993CrossRefPubMedPubMedCentralGoogle Scholar
  49. Liu Z, Lavis LD, Betzig E (2015) Imaging live-cell dynamics and structure at the single-molecule level. Mol Cell 58:644. Scholar
  50. Lorenz WW, McCann RO, Longiaru M, Cormier MJ (1991) Isolation and expression of a cDNA encoding Renilla reniformis luciferase. Proc Natl Acad Sci U S A 88(10):4438–4442. Scholar
  51. Massoud TF, Gambhir SS (2003) Molecular imaging in living subjects: seeing fundamental biological processes in a new light. Genes Dev 17:545–580. Scholar
  52. Matsumoto Y, Jasanoff A (2013) Metalloprotein-based MRI probes. FEBS Lett 587:1021–1029. Scholar
  53. McElroy WD, Hastings JW, Coulombre J, Sonnenfeld V (1953) The mechanism of action of pyrophosphate in firefly luminescence. Arch Biochem Biophys 46(2):399–416. Scholar
  54. Mempel TR, Pittet MJ, Khazaie K et al (2006) Regulatory T cells reversibly suppress cytotoxic T cell function independent of effector differentiation. Immunity 25:129–141. Scholar
  55. Mezzanotte L, van t’ Root M, Karatas H, et al (2017) In vivo molecular bioluminescence imaging: new tools and applications. Trends Biotechnol 35:640–652. Scholar
  56. Minn I, Bar-Shir A, Yarlagadda K et al (2015) Tumor-specific expression and detection of a CEST reporter gene. Magn Reson Med 74:544–549. Scholar
  57. Morin JG, Hastings JW (1971) Energy transfer in a bioluminescent system. J Cell Physiol 77(3):313–318. Scholar
  58. Nafissi N, Slavcev R (2012) Construction and characterization of an in-vivo linear covalently closed DNA vector production system. Microb Cell Fact 11:154. Scholar
  59. Nafissi N, Alqawlaq S, Lee EA et al (2014) DNA ministrings: highly safe and effective gene delivery vectors. Mol Ther Nucleic Acids 3:e165. Scholar
  60. Nagai T, Ibata K, Park ES et al (2002) A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat Biotechnol 20:87–90. Scholar
  61. Nolan T, Hands RE, S a B (2006) Quantification of mRNA using real-time RT-PCR. Nat Protoc 1:1559–1582. Scholar
  62. Overbergh L, Giulietti A, Valckx D, et al (2003) The use of real-time reverse transcriptase PCR for the quantification of cytokine gene expression. J Biomol Tech 14:33–43. PMC2279895Google Scholar
  63. Penheiter AR, Russell SJ, Carlson SK (2012) The sodium iodide symporter (NIS) as an imaging reporter for gene, viral, and cell-based therapies. Curr Gene Ther 12:33–47. Scholar
  64. Permyakov E (1993) Luminescent spectroscopy of proteins. CRC Press, LondonGoogle Scholar
  65. Piatkevich KD, Verkhusha VV (2011) Guide to red fluorescent proteins and biosensors for flow cytometry. Methods Cell Biol.
  66. Pichler BJ, Judenhofer MS, Pfannenberg C (2008) Multimodal imaging approaches: PET/CT and PET/MRI. Handb Exp Pharmacol 185:109–132. Scholar
  67. Rice BW, Cable MD, Nelson MB (2001) In vivo imaging of light-emitting probes. J Biomed Opt 6(4):432–440. Scholar
  68. Richards MP, Poch SM (2002) Quantitative analysis of gene expression by reverse transcription polymerase chain reaction and capillary electrophoresis with laser-induced fluorescence detection. Mol Biotechnol 21:19–37. Scholar
  69. Rizzuto R, Brini M, Pizzo P et al (1995) Chimeric green fluorescent protein as a tool for visualizing subcellular organelles in living cells. Curr Biol 5:635–642. Scholar
  70. Robert S, Lacroix R, Poncelet P et al (2012) High-sensitivity flow cytometry provides access to standardized measurement of small-size microparticles-brief report. Arterioscler Thromb Vasc Biol 32:1054–1058. Scholar
  71. Rodriguez A, Nabi IR, Meighen E (1985) ATP turnover by the fatty acid reductase complex of Photobacterium phosphoreum. Can J Biochem Cell Biol 63(10):1106–1111CrossRefGoogle Scholar
  72. Sadikot RT, Wudel JL, Jansen DE, Debelak JP, Yull FE, Christonan JW et al (2002) Hepatic cryoablation-induced multisystem injury: bioluminescent detection of NF-κB activation in a transgenic mouse model. J Gastrointest Surg 6(2):264–270. Scholar
  73. Sadikot RT, Blackwell TS (2005) Bioluminescence imaging. Proc Am Thorac Soc 2:511–540. doi: Scholar
  74. Sato A, Klaunberg B, Tolwani R (2004) In vivo bioluminescence imaging. Comp Med 54:631–634PubMedGoogle Scholar
  75. Schulze A, Downward J (2001) Navigating gene expression using microarrays—a technology review. Nat Cell Biol 3:E190–E195. Scholar
  76. Seliger HH, McElroy WD (1960) Spectral emission and quantum yield of firefly bioluminescence. Arch Biochem Biophys 88(1):136–141. Scholar
  77. Serganova I, Mayer-Kukuck P, Huang R, Blasberg R (2008) Molecular imaging: reporter gene imaging. Handb Exp Pharmacol 185:167–223. Scholar
  78. Shaner NC, Lin MZ, McKeown MR et al (2008) Improving the photostability of bright monomeric orange and red fluorescent proteins. Nat Methods 5:545–551. Scholar
  79. Shcherbakova DM, Verkhusha VV (2013) Near-infrared fluorescent proteins for multicolor in vivo imaging. Nat Methods 10:751–754. Scholar
  80. Shimomura O (2006) Bioluminescence, Chemical principles and methods, pp 1–29. Scholar
  81. Slonim DK, Yanai I (2009) Getting started in gene expression microarray analysis. PLoS Comput Biol 5:e1000543. Scholar
  82. Soboleski MR, Oaks J, Halford WP (2005) Green fluorescent protein is a quantitative reporter of gene expression in individual eukaryotic cells. Fed Am Soc Exp Biol 19:440–442. Scholar
  83. Stacer AC, Nyati S, Moudgil P, Iyengar R, Luker KE, Rehemtulla A, Luker GD (2013) NanoLuc reporter for dual luciferase imaging in living animals. Mol Imaging 12(7). Scholar
  84. Tangney M, Francis KP (2012) In vivo optical imaging in gene & cell therapy. Curr Gene Ther 12:2–11. Scholar
  85. T.E. Consortium (2011/2016) Standards, guidelines and best practices for RNA-Seq V1.0 (June 2011).
  86. van Roessel P, Brand AH (2002) Imaging into the future: visualizing gene expression and protein interactions with fluorescent proteins. Nat Cell Biol 4:E15–E20. Scholar
  87. Vandsburger MH, Radoul M, Cohen B, Neeman M (2013) MRI reporter genes: applications for imaging of cell survival, proliferation, migration and differentiation. NMR Biomed 26:872–884. Scholar
  88. Velculescu VE, Zhang L, Vogelstein B, Kinzler KW (1995) Serial analysis of gene expression. Science 270:484–487CrossRefPubMedGoogle Scholar
  89. Waerzeggers Y, Monfared P, Viel T et al (2009) Methods to monitor gene therapy with molecular imaging. Methods 48:146–160CrossRefPubMedGoogle Scholar
  90. Waerzeggers Y, Viel T, Schäfers S et al (2013) Positron emission tomography-based molecular imaging. Neuromethods 77:301–327. Scholar
  91. Wang Z, Gerstein M, Snyder M (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10:57–63. Scholar
  92. Wang Z, Wang F, Hida N et al (2015) Design of a functional cyclic HSV1-TK reporter and its application to PET imaging of apoptosis. Nat Protoc 10:807–821. Scholar
  93. Weissleder R, Moore a MU et al (2000) In vivo magnetic resonance imaging of transgene expression. Nat Med 6:351–355. Scholar
  94. Wetterwald A, van der Pluijm G, Que I, Sijmons B, Buijs J, Karperien M, Löwik CW, Gautschi E, Thalmann GN, Cecchini MG (2002) Optical imaging of cancer metastasis to bone marrow: a mouse model of minimal residual disease. Am J Pathol 160(3):1143–1153. Scholar
  95. Widder EA (2010) Bioluminescence in the ocean: origins of biological, chemical, and ecological diversity. Science (80–) 704:704–708. doi: Scholar
  96. Williams RS, Johnston SA, Riedy M et al (1991) Introduction of foreign genes into tissues of living mice by DNA-coated microprojectiles. Proc Natl Acad Sci U S A 88:2726–2730. Scholar
  97. Williams SJ, Schwer C, Krishnarao AS et al (1996) Quantitative competitive polymerase chain reaction: analysis of amplified products of the HIV-1 gag gene by capillary electrophoresis with laser-induced fluorescence detection. Anal Biochem 236:146–152. Scholar
  98. Wong ML, Medrano JF (2005) Real-time PCR for mRNA quantitation. BioTechniques 39(1):75–85CrossRefPubMedGoogle Scholar
  99. Yaghoubi SS, Gambhir SS (2006) PET imaging of herpes simplex virus type 1 thymidine kinase (HSV1-tk) or mutant HSV1-sr39tk reporter gene expression in mice and humans using [18F]FHBG. Nat Protoc 1:3069–3075. Scholar
  100. Yaghoubi SS, Campbell DO, Radu CG, Czernin J (2012) Positron emission tomography reporter genes and reporter probes: gene and cell therapy applications. Theranostics 2:374–391. Scholar
  101. Youn H, Hong KJ (2012) In vivo noninvasive small animal molecular imaging. Osong Public Health Res Perspect 3:48–59. Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  • Roderick Adrian Slavcev
    • 1
    Email author
  • Chi Hong Sum
    • 1
  • Jesse St. Jean
    • 1
  • Haein Huh
    • 1
  • Nafiseh Nafissi
    • 1
  1. 1.University of Waterloo, School of PharmacyWaterlooCanada

Personalised recommendations