Construction and identification of the adenoviral vector with dual reporter gene for multimodality molecular imaging

Abstract

In this study, the recombinant adenovirus (Ad) vector containing dual reporter gene [i.e. human transferrin receptor gene (TFRC) and firefly luciferase reporter gene] was constructed to provide a novel experimental tool for magnetic resonance (MR) and bioluminescence dual-modality molecular imaging. The cDNA of TFRC was amplified by polymerase chain reaction (PCR) and cloned into the multiple cloning site of pShuttle-CMV-CMV-Luciferase vector. After identification by Sfi I digestion and sequencing, pShuttle-TFRC-Luciferase vector and the adenoviral backbone vector (pAdeno) were subjected to homologous recombination. The correct recombinant plasmid was then transfected into 293 packaging cells to produce adenoviral particles and confirmed by PCR. After infection of human colorectal cancer LOVO cells with Ad-TFRC-Luciferase, the expressions of transferrin receptor (TfR) and luciferase protein were detected respectively by Western blotting and bioluminescence imaging in vitro. The results showed that TFRC gene was successfully inserted into the adenoviral shuttle vector carrying luciferase gene. DNA sequence analysis indicated that the TFRC gene sequence in the shuttle plasmid was exactly the same as that reported in GenBank. The recombinant plasmid was identified correct by restriction digestion. Ad-TFRC-Luciferase recombinant adenovirus was constructed successfully, and the virus titer was 1.6×10¹⁰ pfu/mL. Forty-eight h after dual reporter gene transfection, the expressions of TfR and luciferase protein were increased significantly (P<0.01). It was concluded that the recombinant adenovirus vector with dual reporter gene was successfully established, which may be used for invivo tracing target cells in multimodality imaging.

Keywords

adenovirus / transferrin receptor / luciferase / reporter gene / molecular imaging

References

[1] GiladAA, WinnardPT, ZijlPCM, et al.. Developing MR reporter genes: promises and pitfalls. NMR Biomed, 2007, 20: 275-290

[2] WeisslederR, MooreA, MahmoodU, et al.. In vivo magnetic resonance imaging of transgene expression. Nat Med, 2000, 6: 351-355

[3] MooreA, BasilionJP, ChioccaEA, et al.. Measuring transferrin receptor gene expression by NMR imaging. Biochim Biophys Acta, 1998, 1402: 239-249

[4] SerganovaI, Mayer-KukuckP, HuangR, et al.. Molecular imaging: reporter gene imaging. Handbook Exp Pharmacol, 2008, 185(Pt2): 167-223

[5] SadikotRT, BlackwellTS. Bioluminescence: imaging modality for in vitro and in vivo gene expression. Methods Mol Biol, 2008, 477: 383-394

[6] KangJH, ChungJK. Molecular-genetic imaging based on reporter gene expression. J Nucl Med, 2008, 49: 164S-179S

[7] ChenIY, GreveJM, GheysensO, et al.. Comparison of optical bioluminescence reporter gene and superparamagnetic iron oxide MR contrast agent as cell markers for noninvasive imaging of cardiac cell transplantation. Mol Imaging Biol, 2009, 11(3): 178-187

[8] ZhangH, QiaoH, BakkenA, et al.. Utility of dual-modality bioluminescence and MRI in monitoring stem cell survival and impact on post myocardial infarct remodeling. Acad Radiol, 2011, 18(1): 3-12

[9] LiZ, SuzukiY, HuangM, et al.. Comparison of reporter gene and iron particle labeling for tracking fate of human embryonic stem cells and differentiated endothelial cells in living subjects. Stem Cells, 2008, 26: 864-873

[10] SongHT, JordanEK, LewisBK, et al.. Rat model of metastatic breast cancer monitored by MRI at 3 tesla and bioluminescence imaging with histological correlation. J Transl Med, 2009, 7: 88

[11] BlackPC, ShettyA, BrownGA, et al.. Validating bladder cancer xenograft bioluminescence with magnetic resonance imaging: the significance of hypoxia and necrosis. BJU Int, 2010, 106(11): 1799-1804

[12] WeisslederR, MahmoodU. Molecular imaging. Radiology, 2001, 219: 316-333

[13] JafferFA, WeisslederR. Molecular imaging in the clinical arena. JAMA, 2005, 293(7): 855-862

[14] MassoudTF, GambhirSS. Molecular imaging in living subjects: seeing fundamental biological processes in a new light. Genes Dev, 2003, 17(5): 545-580

[15] ThakurM, LentleBC. Report of a summit on molecular imaging. Radiology, 2005, 236(3): 753-755

[16] PyszMA, GambhirSS, WillmannJK. Molecular imaging: current status and emerging strategies. Clin Radiol, 2010, 65(7): 500-516

[17] GoertzenAL, MeadorsAK, SilvermanRW, et al.. Simultaneous molecular and anatomical imaging of the mouse in vivo. Phys Med Biol, 2002, 47(24): 4315-4328

[18] TownsendDW, BeyerT, BlodgettTM. PET/CT scanners: a hardware approach to image fusion. Semin Nucl Med, 2003, 33(3): 193-204

[19] LouieA. Multimodality imaging probes: design and challenges. Chem Rev, 2010, 110(5): 3146-3195

[20] XieJ, ChenK, HuangJ, et al.. PET/NIRF/MRI triple functional iron oxide nanoparticles. Biomaterials, 2010, 31(11): 3016-3022

[21] IyerM, SatoM, JohnsonM, et al.. Applications of molecular imaging in cancer gene therapy. Curr Gene Ther, 2005, 5(6): 607-618

[22] LiuT, ZhouH, XiaR, et al.. Tracking tumor cells in lymphatics in a mice xenograft model by magnetic resonance imaging. Mol Imaging, 2012, 11(6): 451-460

[23] WangK, ShenB, HuangT, et al.. MR reporter gene imaging of endostatin expression and therapy. Mol Imaging Biol, 2010, 12(5): 520-529

[24] AungW, HasegawaS, Koshikawa-YanoM, et al.. Visualization of in vivo electroporation-mediated transgene expression in experimental tumors by optical and magnetic resonance imaging. Gene Therapy, 2009, 16: 830-839

[25] BenihoudK, YehP, PerricaudetM. Adenovirus vectors for gene delivery. Curr Opin Biotechnol, 1999, 10(5): 440-447

[26] HeTC, ZhouS, CostaLT, et al.. A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci, 1998, 95: 2509-2514