Wang Yi-Fan, Liu Ting, Guo Yu-Lin, Gao Fa-Bao
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.
J Huazhong Univ Sci Technolog Med Sci. 2013 Aug;33(4):600-605. doi: 10.1007/s11596-013-1165-0. Epub 2013 Aug 1.
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.
在本研究中，构建了包含双报告基因[即人转铁蛋白受体基因（TFRC）和萤火虫荧光素酶报告基因]的重组腺病毒（Ad）载体，为磁共振（MR）和生物发光双模态分子成像提供了一种新型实验工具。通过聚合酶链反应（PCR）扩增TFRC的cDNA，并将其克隆到pShuttle-CMV-CMV-荧光素酶载体的多克隆位点。经Sfi I酶切鉴定和测序后，将pShuttle-TFRC-荧光素酶载体与腺病毒骨架载体（pAdeno）进行同源重组。然后将正确的重组质粒转染至293包装细胞中以产生腺病毒颗粒，并通过PCR进行确认。用Ad-TFRC-荧光素酶感染人结肠癌细胞LOVO后，分别通过蛋白质免疫印迹法和体外生物发光成像检测转铁蛋白受体（TfR）和荧光素酶蛋白的表达。结果表明，TFRC基因成功插入到携带荧光素酶基因的腺病毒穿梭载体中。DNA序列分析表明，穿梭质粒中的TFRC基因序列与GenBank中报道的序列完全相同。通过限制性酶切鉴定重组质粒正确。成功构建了Ad-TFRC-荧光素酶重组腺病毒，病毒滴度为1.6×10(10) pfu/mL。双报告基因转染48小时后，TfR和荧光素酶蛋白的表达显著增加（P<0.01）。结论是成功建立了具有双报告基因的重组腺病毒载体，可用于多模态成像中体内追踪靶细胞。
