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首页> 外文期刊>Experimental and therapeutic medicine >Feasibility of using a dual-promoter recombinant baculovirus vector to coexpress EGFP and GDNF in mammalian cells
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Feasibility of using a dual-promoter recombinant baculovirus vector to coexpress EGFP and GDNF in mammalian cells

机译：使用双启动子重组杆状病毒载体在哺乳动物细胞中共表达EGFP和GDNF的可行性

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Vectors that are capable of coexpressing two or more exogenous genes for in vitro and in vivo 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 trans- fected 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.
机译：能够共表达两个或更多个外源基因用于体外和体内基因递送的载体正在被越来越多的研究。本研究的目的是探讨使用pFastBac？的可行性。在两个具有相反方向的巨细胞病毒（CMV）启动子的控制下，双重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）。在这项研究中，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胎肾（HEK）293T细胞中。重组病毒Bac Dual-CMV-EGFP-CMV-GDNF是使用Bac-to-Bac杆状病毒表达系统生成的，可用于转导HeLa细胞。免疫荧光检测转染或转导的哺乳动物细胞中EGFP和GDNF的共表达，而蛋白质印迹分析用于确认转导的HeLa细胞中GDNF的表达。构建了重组质粒，并成功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细胞和单个转染的HeLa细胞中同时表达。 Western印迹分析表明GDNF在转导的细胞中准确表达。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用作载体。

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