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A novel triple-modality reporter gene for whole-body fluorescent, bioluminescent, and nuclear noninvasive imaging

  • Molecular Imaging
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Abstract

Two genetic reporter systems were developed for multimodality reporter gene imaging of different molecular-genetic processes using fluorescence, bioluminescence (BLI), and nuclear imaging techniques. The eGFP cDNA was fused at the N-terminus with HSV1-tk cDNA bearing a nuclear export signal from MAPKK (NES-HSV1-tk) or with truncation at the N-terminus of the first 45 amino acids (Δ45HSV1-tk) and with firefly luciferase at the C-terminus. A single fusion protein with three functional subunits is formed following transcription and translation from a single open reading frame. The NES-TGL (NES-TGL) or Δ45HSV1-tk/GFP/luciferase (Δ45-TGL) triple-fusion gene cDNAs were cloned into a MoMLV-based retrovirus, which was used for transduction of U87 human glioma cells. The integrity, fluorescence, bioluminescence, and enzymatic activity of the TGL reporter proteins were assessed in vitro. The predicted molecular weight of the fusion proteins (~130 kDa) was confirmed by western blot. The U87-NES-TGL and U87-Δ45-TGL cells had cytoplasmic green fluorescence. The in vitro BLI was 7- and 13-fold higher in U87-NES-TGL and U87-Δ45-TGL cells compared to nontransduced control cells. The Ki of 14C-FIAU was 0.49±0.02, 0.51±0.03, and 0.003±0.001 ml/min/g in U87-NES-TGL, U87-Δ45-TGL, and wild-type U87 cells, respectively. Multimodality in vivo imaging studies were performed in nu/nu mice bearing multiple s.c. xenografts established from U87-NES-TGL, U87-Δ45-TGL, and wild-type U87 cells. BLI was performed after administration of d-luciferin (150 mg/kg i.v.). Gamma camera or PET imaging was conducted at 2 h after i.v. administration of [131I]FIAU (7.4 MBq/animal) or [124I]FIAU (7.4 MBq/animal), respectively. Whole-body fluorescence imaging was performed in parallel with the BLI and radiotracer imaging studies. In vivo BLI and gamma camera imaging showed specific localization of luminescence and radioactivity to the TGL transduced xenografts with background levels of activity in the wild-type xenografts. Tissue sampling yielded values of 0.47%±0.08%, 0.86%±0.06%, and 0.03%±0.01%dose/g [131I]FIAU in U87-NES-TGL, U87-Δ45-TGL, and U87 xenografts, respectively. The TGL triple-fusion reporter gene preserves the functional activity of its subunits and is very effective for multimodality imaging. It provides for the seamless transition from fluorescence microscopy and FACS to whole-body bioluminescence imaging, to nuclear (PET, SPET, gamma camera) imaging, and back to in situ fluorescence image analysis.

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References

  1. Blasberg RG, Tjuvajev JG. Herpes simplex virus thymidine kinase as a marker/reporter gene for PET imaging of gene therapy. Q J Nucl Med 1999; 43:163–169.

    CAS  PubMed  Google Scholar 

  2. Gambhir SS, Herschman HR, Cherry SR, Barrio JR, Satyamurthy N, Toyokuni T, Phelps ME, Larson SM, Balatoni J, Finn R, Sadelain M, Tjuvajev J, Blasberg R. Imaging transgene expression with radionuclide imaging technologies. Neoplasia 2000; 2:118–138.

    CAS  PubMed  Google Scholar 

  3. Gelovani Tjuvajev J, Blasberg R. In vivo imaging of molecular-genetic targets for cancer therapy. Cancer Cell 2003; 3:327–332.

    PubMed  Google Scholar 

  4. Doubrovin M, Ponomarev V, Beresten T, Balatoni J, Bornmann W, Finn R, Humm J, Larson S, Sadelain M, Blasberg R, Gelovani Tjuvajev J. Imaging transcriptional regulation of p53-dependent genes with positron emission tomography in vivo. Proc Natl Acad Sci U S A 2001; 98:9300–9305.

    Article  CAS  PubMed  Google Scholar 

  5. Ponomarev V, Doubrovin M, Lyddane C, Beresten T, Balatoni J, Bornman W, Finn R, Akhurst T, Larson S, Blasberg R, Sadelain M, Tjuvajev JG. Imaging TCR-dependent NFAT-mediated T-cell activation with positron emission tomography in vivo. Neoplasia 2001; 3:480–488.

    Article  CAS  PubMed  Google Scholar 

  6. Adams JY, Johnson M, Sato M, Berger F, Gambhir SS, Carey M, Iruela-Arispe ML, Wu L. Visualization of advanced human prostate cancer lesions in living mice by a targeted gene transfer vector and optical imaging. Nat Med 2002; 8:891–897.

    CAS  PubMed  Google Scholar 

  7. Luker GD, Sharma V, Pica CM, Dahlheimer JL, Li W, Ochesky J, Ryan CE, Piwnica-Worms H, Piwnica-Worms D. Noninvasive imaging of protein-protein interactions in living animals. Proc Natl Acad Sci U S A 2002; 99:6961–6966.

    Article  CAS  PubMed  Google Scholar 

  8. Serganova I, Ponomarev V, Doubrovin M, Ageyeva L, Beresten T, Soghomonian S, Balatoni J, Finn R, Blasberg R, Gelovani Tjuvajev JG. Successful generation of reporter system for non-invasive imaging of the TGFb signal transduction pathway activity with positron emission tomography In Vivo. ASGT 5th Annual Meeting, Boston, June 5–9, 2002.

  9. Resnicoff M, Tjuvajev J, Rotman HL, Abraham D, Curtis M, Aiken R, Baserga R. Regression of C6 rat brain tumors by cells expressing an antisense insulin-like growth factor I receptor RNA. J Exp Ther Oncol 1996; 1:385–389.

    CAS  PubMed  Google Scholar 

  10. De A, Lewis XZ, Gambhir SS. Noninvasive imaging of lentiviral-mediated reporter gene expression in living mice. Mol Ther 2003; 7:681–691.

    Article  CAS  PubMed  Google Scholar 

  11. Gambhir SS, Barrio JR, Wu L, Iyer M, Namavari M, Satyamurthy N, Bauer E, Parrish C, MacLaren DC, Borghei AR, Green LA, Sharfstein S, Berk AJ, Cherry SR, Phelps ME, Herschman HR. Imaging of adenoviral-directed herpes simplex virus type 1 thymidine kinase reporter gene expression in mice with radiolabeled ganciclovir. J Nucl Med 1998; 39:2003–2011.

    CAS  PubMed  Google Scholar 

  12. Bennett JJ, Tjuvajev J, Johnson P, Doubrovin M, Akhurst T, Malholtra S, Hackman T, Balatoni J, Finn R, Larson SM, Federoff H, Blasberg R, Fong Y. Positron emission tomography imaging for herpes virus infection: implications for oncolytic viral treatments of cancer. Nat Med 2001; 7:859–863.

    Article  CAS  PubMed  Google Scholar 

  13. Koehne G, Doubrovin M, Doubrovina E, Zanzonico P, Gallardo HF, Ivanova A, Balatoni J, Teruya-Feldstein J, Heller G, May C, Ponomarev V, Ruan S, Finn R, Blasberg RG, Bornmann W, Riviere I, Sadelain M, O’Reilly RJ, Larson SM, Gelovani Tjuvajev JG. Serial in vivo imaging of the targeted migration of human HSV-TK-transduced antigen-specific lymphocytes. Nat Biotechnol 2003; 21:405-413.

    Article  CAS  PubMed  Google Scholar 

  14. Dubey P, Su H, Adonai N, Du S, Rosato A, Braun J, Gambhir SS, Witte ON. Quantitative imaging of the T cell antitumor response by positron-emission tomography. Proc Natl Acad Sci U S A 2003; 100:1232–1237.

    Article  CAS  PubMed  Google Scholar 

  15. Tjuvajev JG, Stockhammer G, Desai R, Uehara H, Watanabe K, Gansbacher B, Blasberg RG. Imaging the expression of transfected genes in vivo. Cancer Res 1995; 55:6126–6132.

    CAS  PubMed  Google Scholar 

  16. Jacobs A, Tjuvajev JG, Dubrovin M, Akhurst T, Balatoni J, Beattie B, Joshi R, Finn R, Larson SM, Herrlinger U, Pechan PA, Chiocca EA, Breakefield XO, Blasberg RG. Positron emission tomography-based imaging of transgene expression mediated by replication-conditional, oncolytic herpes simplex virus type 1 mutant vectors in vivo. Cancer Res 2001; 61:2983–2995.

    CAS  PubMed  Google Scholar 

  17. Tjuvajev JG, Avril N, Oku T, Sasajima T, Miyagawa T, Joshi R, Safer M, Beattie B, DiResta G, Daghighian F, Augensen F, Koutcher J, Zweit J, Humm J, Larson SM, Finn R, Blasberg R. Imaging herpes virus thymidine kinase gene transfer and expression by positron emission tomography. Cancer Res 1998; 58:4333–4341.

    CAS  PubMed  Google Scholar 

  18. Ponomarev V, Doubrovin M, Serganova I, Beresten T, Vider J, Shavrin A, Ageeva L, Balatoni J, Blasberg R, Gelovani Tjuvajev J. Cytoplasmically retargeted HSV1-tk/GFP reporter gene mutants for optimization of non-invasive molecular-genetic imaging. Neoplasia 2003; 5:245–254.

    CAS  PubMed  Google Scholar 

  19. Doubrovin M, Ponomarev V, Serganova I, Soghomonian S, Myagawa T, Beresten T, Ageyeva L, Sadelain M, Koutcher J, Blasberg RJ. Tjuvajev JG. Development of a new reporter gene system—dsRed/xanthine phosphoribosyltransferase-xanthine for molecular imaging of processes behind the intact blood-brain barrier. Mol Imaging 2003; 2:93–112.

    Article  CAS  PubMed  Google Scholar 

  20. Dubrovin M, Ponomarev V, Beresten T, Matei C, Koutcher J, Tjuvajev J. In vivo19F nuclear magnetic resonance measurements of enhanced 5FU conversion to fluoronucleotides after UPRT gene transduction [abstract]. 3rd Annual Meeting of the ASGT, Denver, CO, May 31–June 4, 2000.

  21. Ponomarev V, Doubrovin M, Serganova I, Ageyeva L, Beresten T, Sagomonyan S, Balatoni J, Finn R, Blasberg R, Gelovani Tjuvajev JG. Human thymidine kinase type 2—a novel non-immunogenic reporter gene for non-invasive imaging in humans. AACR meeting: Molecular imaging in cancer, Orlando, Fl, January 23–27, 2002.

  22. Liang Q, Satyamurthy N, Barrio JR, Toyokuni T, Phelps MP, Gambhir SS, Herschman HR. Noninvasive, quantitative imaging in living animals of a mutant dopamine D2 receptor reporter gene in which ligand binding is uncoupled from signal transduction. Gene Ther 2001; 8:1490–1498.

    CAS  PubMed  Google Scholar 

  23. Rogers BE, Zinn KR, Buchsbaum DJ. Gene transfer strategies for improving radiolabeled peptide imaging and therapy. Q J Nucl Med 2000; 3:208–223.

    Google Scholar 

  24. Brust P, Haubner R, Friedrich A, Scheunemann M, Anton M, Koufaki ON, Hauses M, Noll S, Noll B, Haberkorn U, Schackert G, Schackert HK, Avril N, Johannsen B. Comparison of [18F]FHPG and [124/125I]FIAU for imaging herpes simplex virus type 1 thymidine kinase gene expression. Eur J Nucl Med 2001; 28:721–729.

    CAS  PubMed  Google Scholar 

  25. Stegman LD, Rehemtulla A, Beattie B, Kievit E, Lawrence TS, Blasberg RG, Tjuvajev JG, Ross BD. Noninvasive quantitation of cytosine deaminase transgene expression in human tumor xenografts with in vivo magnetic resonance spectroscopy. Proc Natl Acad Sci U S A 1999; 96:9821–9826.

    CAS  PubMed  Google Scholar 

  26. Hackman T, Doubrovin M, Balatoni J, Beresten T, Ponomarev V, Beattie B, Finn R, Bornmann W, Blasberg R, Gelovani Tjuvajev J. Imaging expression of cytosine deaminase-herpes virus thymidine kinase fusion gene (CD/TK) expression with [124I]FIAU and PET. Mol Imaging 2002; 1:36–42.

    Article  CAS  PubMed  Google Scholar 

  27. Hadjantonakis AK, Nagy A. The color of mice: in the light of GFP-variant reporters. Histochem Cell Biol 2001; 115:49–58.

    CAS  PubMed  Google Scholar 

  28. Falk MM, Lauf U. High resolution, fluorescence deconvolution microscopy and tagging with the autofluorescent tracers CFP, GFP, and YFP to study the structural composition of gap junctions in living cells. Microsc Res Tech 2001; 52:251–262.

    CAS  PubMed  Google Scholar 

  29. Ellenberg J, Lippincott-Schwartz J, Presley JF. Dual-colour imaging with GFP variants. Trends Cell Biol 1999; 9:52–56.

    CAS  PubMed  Google Scholar 

  30. Contag CH, Jenkins D, Contag PR, Negrin RS. Use of reporter genes for optical measurements of neoplastic disease in vivo. Neoplasia 2000; 2:41–52.

    CAS  PubMed  Google Scholar 

  31. Levy JP, Muldoon RR, Zolotukhin S, Link CJ Jr. Retroviral transfer and expression of a humanized, red-shifted green fluorescent protein gene into human tumor cells. Nat Biotechnol 1996; 14:610–614.

    CAS  PubMed  Google Scholar 

  32. Lalwani AK, Han JJ, Walsh BJ, Zolotukhin S, Muzyczka N, Mhatre AN. Green fluorescent protein as a reporter for gene transfer studies in the cochlea. Hear Res 1997; 114:139–147.

    Article  CAS  PubMed  Google Scholar 

  33. Campbell RE, Tour O, Palmer AE, Steinbach PA, Baird GS, Zacharias DA, Tsien RY. A monomeric red fluorescent protein. Proc Natl Acad Sci U S A 2002; 99:7877–7882.

    Article  CAS  PubMed  Google Scholar 

  34. Mathieu S, El-Battari A. Monitoring E-selectin-mediated adhesion using green and red fluorescent proteins. J Immunol Methods 2003; 272:81–92.

    Article  CAS  PubMed  Google Scholar 

  35. Bhaumik S, Gambhir SS. Optical imaging of Renilla luciferase reporter gene expression in living mice. Proc Natl Acad Sci U S A 2002; 99:377–382.

    CAS  PubMed  Google Scholar 

  36. Ray P, Bauer E, Iyer M, Barrio JR, Satyamurthy N, Phelps ME, Herschman HR, Gambhir SS. Monitoring gene therapy with reporter gene imaging. Semin Nucl Med 2001; 31:312–320.

    CAS  PubMed  Google Scholar 

  37. Beyer T, Townsend DW, Brun T, Kinahan PE, Charron M, Roddy R, Jerin J, Young J, Byars L, Nutt R. A combined PET/CT scanner for clinical oncology. J Nucl Med 2000; 41:1369–1379.

    CAS  PubMed  Google Scholar 

  38. Jacobs A, Dubrovin M, Hewett J, Sena-Esteves M, Tan CW, Slack M, Sadelain M, Breakefield XO, Tjuvajev JG. Functional coexpression of HSV-1 thymidine kinase and green fluorescent protein: implications for noninvasive imaging of transgene expression. Neoplasia 1999; 1:154–161.

    Article  CAS  PubMed  Google Scholar 

  39. Wang Y, Yu YA, Shabahang S, Wang G, Szalay AA. Renilla luciferase-Aequorea GFP (Ruc-GFP) fusion protein, a novel dual reporter for real-time imaging of gene expression in cell cultures and in live animals. Mol Genet Genomics 2002; 268:160–168.

    Article  CAS  PubMed  Google Scholar 

  40. Ray P, Wu AM, Gambhir SS. Optical bioluminescence and positron emission tomography imaging of a novel fusion reporter gene in tumor xenografts of living mice. Cancer Res 2003; 63:1160–1165.

    CAS  PubMed  Google Scholar 

  41. Ponomarev V, Doubrovin M, Serganova I, Ageyeva L, Beresten T, Sagomonyan S, Balatoni J, Finn R, Blasberg R, Gelovani Tjuvajev, JG. A novel triple modality reporter gene for whole body fluorescent, bioluminescent and nuclear non-invasive imaging. ASGT 6th Annual Meeting, Washington DC, June 4–8 2003.

  42. Gallardo HF, Tan C, Ory D, Sadelain M. Recombinant retroviruses pseudotyped with the vesicular stomatitis virus G glycoprotein mediate both stable gene transfer and pseudotransduction in human peripheral blood lymphocytes. Blood 1997; 90:952–957.

    CAS  PubMed  Google Scholar 

  43. Miyagawa T, Oku T, Uehara H, Desai R, Beattie B, Tjuvajev J, Blasberg R. “Facilitated” amino acid transport is upregulated in brain tumors. J Cereb Blood Flow Metab 1998; 18:500–509.

    Article  CAS  PubMed  Google Scholar 

  44. Tjuvajev JG, Joshi A, Callegari J, Lindsley L, Joshi R, Balatoni J, Finn R, Larson SM, Sadelain M, Blasberg RG. A general approach to the non-invasive imaging of transgenes using cis-linked herpes simplex virus thymidine kinase. Neoplasia 1999; 1:315–320.

    CAS  PubMed  Google Scholar 

  45. Ivanova A, Ponomarev V, Dubrovin M, Ageyeva L, Serganova I, Vider E, Soghomonian S, Balatoni J, Finn R, Blasberg R, Gelovani Tjuvajev, J. A novel retroviral vector for transfection with multiple reporter genes for multi-modality imaging in vivo. Society of Molecular Imaging 1st Annual Meeting, Boston, MA, August 24–26, 2002.

  46. Borman A, Le Mercier P, Girard M, Kean KM. Comparison of picornaviral IRES-driven internal initiation of translation in cultured cells of different origins. Nucleic Acids Res 1997; 25:925–932.

    Article  CAS  PubMed  Google Scholar 

  47. Robbins P, Ghivizzani SC. Viral vectors for gene therapy. Pharmacol Ther 1998; 80:35–47.

    CAS  PubMed  Google Scholar 

  48. Paquin A, Jaalouk DE, Galipeau J. Retrovector encoding a green fluorescent protein-herpes simplex virus thymidine kinase fusion protein serves as a versatile suicide/reporter for cell and gene therapy applications. Hum Gene Ther 2001; 12:13–23.

    Article  CAS  PubMed  Google Scholar 

  49. Conti E, Franks NP, Brick P. Crystal structure of firefly luciferase throws light on a superfamily of adenylate-forming enzymes. Structure 1996; 5:287–298.

    Google Scholar 

  50. Salomon B, Maury S, Loubiere L, Caruso M, Onclercq R, Klatzmann D. A truncated herpes simplex virus thymidine kinase phosphorylates thymidine and nucleoside analogs and does not cause sterility in transgenic mice. Mol Cell Biol 1995; 15:5322–5328.

    CAS  PubMed  Google Scholar 

  51. Cohen JL, Boyer O, Salomon B, Onclerco R, Depetris D, Lejeune L, Dubus-Bonnet V, Bruel S, Charlotte F, Mattei MG, Klatzmann D. Fertile homozygous transgenic mice expressing a functional truncated herpes simplex thymidine kinase delta TK gene. Transgenic Res 1998; 7:321–330.

    Article  CAS  PubMed  Google Scholar 

  52. Cowsill C, Southgate TD, Morrissey G, Dewey RA, Morelli AE, Maleniak TC, Forrest Z, Klatzmann D, Wilkinson GW, Lowenstein PR, Castro MG. Central nervous system toxicity of two adenoviral vectors encoding variants of the herpes simplex virus type 1 thymidine kinase: reduced cytotoxicity of a truncated HSV1-TK. Gene Ther 2000; 7:679–685.

    Article  CAS  PubMed  Google Scholar 

  53. Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y. ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett 1997; 407:313–319.

    CAS  PubMed  Google Scholar 

  54. DiLella A, Hope DA, Chen H, Trumbauer M, Schwartz RJ, Smith RG. Utility of firefly luciferase as a reporter gene for promoter activity in transgenic mice. Nucleic Acids Res 1988; 16:4159.

    CAS  PubMed  Google Scholar 

  55. Green L, Yap C, Nguyen N, Barrio J, Namavari M, Satyamurthy N, Phelps M, Sandgren E, Herschman H, Gambhir S. Indirect monitoring of endogenous gene expression by positron emission tomography (PET) imaging of reporter gene expression in transgenic mice. Mol Imaging Biol 2002; 4:71–81.

    Article  Google Scholar 

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  1. Juri Gelovani Tjuvajev

    Present address: MD Anderson Cancer Center, 1515 Holcombe Road, Box 0057, Houston, TX 77030, USA

Authors and Affiliations

  1. Department of Radiology, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA

    Vladimir Ponomarev, Jelena Vider, Aleksander Shavrin, Ludmila Ageyeva, Vilia Tourkova & Juri Gelovani Tjuvajev

  2. Department of Neurology, Memorial Sloan-Kettering Cancer Center, New York, USA

    Michael Doubrovin, Inna Serganova, Tatiana Beresten, Anna Ivanova & Ronald Blasberg

  3. Radiochemistry/Cyclotron Core Facility, Memorial Sloan-Kettering Cancer Center, New York, USA

    Julius Balatoni

  4. Organic Chemistry Synthesis Core Facility, Memorial Sloan-Kettering Cancer Center, New York, USA

    William Bornmann

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  1. Vladimir Ponomarev
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Ponomarev, V., Doubrovin, M., Serganova, I. et al. A novel triple-modality reporter gene for whole-body fluorescent, bioluminescent, and nuclear noninvasive imaging. Eur J Nucl Med Mol Imaging 31, 740–751 (2004). https://doi.org/10.1007/s00259-003-1441-5

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