Wang Jianzhang, Wang Jun, Cai Changping, Wang Shili, Liu Shuai, Shi Shuo, Zhang Yifan, Li Biao
Department of Otolaryngology, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, P.R. China.
Department of Nuclear Medicine, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, P.R. China.
Exp Ther Med. 2014 Jun;7(6):1549-1554. doi: 10.3892/etm.2014.1655. Epub 2014 Mar 31.
Vectors that are capable of coexpressing two or more exogenous genes for and gene delivery are being increasingly studied. The aim of the present study was to explore the feasibility of using the pFastBac™ Dual vector, under the control of two cytomegalovirus (CMV) promoters with opposite directions, to coexpress enhanced green fluorescent protein (EGFP) and glial cell line-derived neurotrophic factor (GDNF) in the same mammalian cell. In the study, two promoters in the pFastBac Dual vector were replaced with CMV-EGFP and CMV-GDNF, whose directions were consistent with the initial directions. The pFastBac Dual-CMV-EGFP-CMV-GDNF plasmid was constructed and then transfected into human embryonic kidney (HEK) 293T cells. The recombinant virus, Bac Dual-CMV-EGFP-CMV-GDNF, was generated with the Bac-to-Bac Baculovirus Expression system and used to transduce HeLa cells. Immunofluorescence was applied to examine the coexpression of EGFP and GDNF in transfected or transduced mammalian cells, while western blot analysis was used to confirm the expression of GDNF in transduced HeLa cells. The recombinant plasmid was constructed and the recombinant baculovirus was successfully generated. Immunofluorescence observations demonstrated that EGFP and GDNF were simultaneously expressed in the same transfected HEK 293T cell and in a single transduced HeLa cell. Western blot analysis revealed that GDNF was expressed accurately in the transduced cells. Therefore, the pFastBac Dual vector is an efficient gene transfer vector that is able to coexpress two target proteins in mammalian cells and serve as a platform for combining reporter or/and therapy genes used in molecular imaging and dual-gene therapy. Thus, the current study presents a new coexpression strategy for dual-gene delivery and .
能够共表达两个或更多外源基因用于基因递送的载体正受到越来越多的研究。本研究的目的是探讨在两个方向相反的巨细胞病毒（CMV）启动子控制下，使用pFastBac™ Dual载体在同一哺乳动物细胞中共表达增强型绿色荧光蛋白（EGFP）和胶质细胞源性神经营养因子（GDNF）的可行性。在该研究中，pFastBac Dual载体中的两个启动子被CMV-EGFP和CMV-GDNF取代，其方向与初始方向一致。构建了pFastBac Dual-CMV-EGFP-CMV-GDNF质粒，然后将其转染到人胚肾（HEK）293T细胞中。利用Bac-to-Bac杆状病毒表达系统产生重组病毒Bac Dual-CMV-EGFP-CMV-GDNF，并用于转导HeLa细胞。应用免疫荧光法检测转染或转导的哺乳动物细胞中EGFP和GDNF的共表达情况，同时采用蛋白质印迹分析来确认转导的HeLa细胞中GDNF的表达。成功构建了重组质粒并产生了重组杆状病毒。免疫荧光观察表明，EGFP和GDNF在同一转染的HEK 293T细胞和单个转导的HeLa细胞中同时表达。蛋白质印迹分析显示GDNF在转导细胞中准确表达。因此，pFastBac Dual载体是一种有效的基因转移载体，能够在哺乳动物细胞中共表达两种靶蛋白，并可作为用于分子成像和双基因治疗的报告基因或/和治疗基因组合的平台。因此，本研究提出了一种用于双基因递送的新的共表达策略。
