Construction And Identification Of Adenoviral Vector With Dual Reporter Gene For Multimodality Molecular Imaging

Objective Recombinant adenovirus (Ad) vector containing dual reporter gene thatconsists of human transferrin receptor gene (TFRC) and firefly luciferase gene wasconstructed to provide a novel experimental tool for magnetic resonance (MR) andbioluminescence dual-modality molecular imaging.Methods The luciferase gene was amplified by polymerase chain reaction (PCR) frompsiCHECK-2plasmid and cloned into the adenoviral shuttle vector (pShuttle-CMV). It wasconfirmed by Nhe Ⅰ/Xba Ⅰ digestion and sequencing. Then the cDNA of TFRC wasamplified with PCR and cloned into the multiple cloning site of pShuttle-CMV-CMV-Lucvector. After identification by Sfi I digestion and sequencing, pShuttle-TFRC-Luc vector andthe adenoviral backbone vector (pAdeno) were homologous recombined. The correctrecombinant plasmid was then transfected into293packaging cells to produce adenoviralparticles and confirmed by PCR. The virus titer was detected. After infecting humancolorectal cancer LOVO cells with Ad-TFRC-Luc, the expressions of transferrin receptor(TfR) and luciferase protein were detected respectively by western blot, Q-PCR andbioluminescence imaging in vitro.Results TFRC gene was successfully inserted into the adenoviral shuttle vectorcarrying luciferase gene. DNA sequence analysis indicated that TFRC gene sequence in theshuttle plasmid was exactly the same as that reported in GenBank. The recombinant plasmidwas identified correct by restriction digestion. Ad-TFRC-Luc recombinant adenovirus wasconstructed successfully, and the virus titer was1.6×1010pfu/ml.48h after dual reporter genetransfection, the expressions of TfR and luciferase protein were increased significantly (P<0.01).Conclusions Ad-TFRC-Luc recombinant adenovirus was constructed successfully.The recombinant adenovirus vector with dual reporter gene might be useful in multimodalityimaging to trace the tumor growth, gene therapy and transplanted stem cells in vivo.