title: 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 1.6×10(10) 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 in vivo tracing target cells in multimodality imaging.
authors.affiliation: Department of Radiology, Molecular Imaging Laboratory, West China Hospital, Sichuan University, Chengdu, 610041, China.|Department of Radiology, General Hospital of Ningxia Medical University, Yinchuan, 750004, China.
authors.forename: Yi-Fan
authors.initials: YF
authors.lastname: Wang
authors.affiliation: Department of Radiology, Molecular Imaging Laboratory, West China Hospital, Sichuan University, Chengdu, 610041, China.
authors.forename: Ting
authors.initials: T
authors.lastname: Liu
authors.affiliation: Department of Radiology, General Hospital of Ningxia Medical University, Yinchuan, 750004, China. guoyulin66@163.com.
authors.forename: Yu-Lin
authors.initials: YL
authors.lastname: Guo
authors.affiliation: Department of Radiology, Molecular Imaging Laboratory, West China Hospital, Sichuan University, Chengdu, 610041, China. gaofabao@yahoo.com.
authors.forename: Fa-Bao
authors.initials: FB
authors.lastname: Gao
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics*; D057054:Molecular Imaging / Q000379:methods
references.citation: Genes Dev. 2003 Mar 1;17(5):545-80
references.pmid: 12629038
references.citation: Mol Imaging. 2012 Nov-Dec;11(6):451-60
references.pmid: 23084246
references.citation: Nat Med. 2000 Mar;6(3):351-5
references.pmid: 10700241
references.citation: Clin Radiol. 2010 Jul;65(7):500-16
references.pmid: 20541650
references.citation: Handb Exp Pharmacol. 2008;(185 Pt 2):167-223
references.pmid: 18626603
references.citation: Chem Rev. 2010 May 12;110(5):3146-95
references.pmid: 20225900
references.citation: Acad Radiol. 2011 Jan;18(1):3-12
references.pmid: 21145025
references.citation: J Nucl Med. 2008 Jun;49 Suppl 2:164S-79S
references.pmid: 18523072
references.citation: Stem Cells. 2008 Apr;26(4):864-73
references.pmid: 18218820
references.citation: Radiology. 2001 May;219(2):316-33
references.pmid: 11323453
references.citation: JAMA. 2005 Feb 16;293(7):855-62
references.pmid: 15713776
references.citation: Mol Imaging Biol. 2010 Oct;12(5):520-9
references.pmid: 19957205
references.citation: Proc Natl Acad Sci U S A. 1998 Mar 3;95(5):2509-14
references.pmid: 9482916
references.citation: Biochim Biophys Acta. 1998 Apr 24;1402(3):239-49
references.pmid: 9606982
references.citation: NMR Biomed. 2007 May;20(3):275-90
references.pmid: 17451181
references.citation: Mol Imaging Biol. 2009 May-Jun;11(3):178-87
references.pmid: 19034584
references.citation: Phys Med Biol. 2002 Dec 21;47(24):4315-28
references.pmid: 12539974
references.citation: Semin Nucl Med. 2003 Jul;33(3):193-204
references.pmid: 12931321
references.citation: Curr Opin Biotechnol. 1999 Oct;10(5):440-7
references.pmid: 10508634
references.citation: Radiology. 2005 Sep;236(3):753-5
references.pmid: 16118158
references.citation: BJU Int. 2010 Dec;106(11):1799-804
references.pmid: 20500508
references.citation: Curr Gene Ther. 2005 Dec;5(6):607-18
references.pmid: 16457650
references.citation: Gene Ther. 2009 Jul;16(7):830-9
references.pmid: 19458649
references.citation: Biomaterials. 2010 Apr;31(11):3016-22
references.pmid: 20092887
references.citation: J Transl Med. 2009 Oct 20;7:88
references.pmid: 19840404
references.citation: Methods Mol Biol. 2008;477:383-94
references.pmid: 19082962