title: Feasibility of using a dual-promoter recombinant baculovirus vector to coexpress EGFP and GDNF in mammalian cells.
abstract: Vectors that are capable of coexpressing two or more exogenous genes forin vitroandin vivogene 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 deliveryin vitroandin vivo.
authors.affiliation: Department of Otolaryngology, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, P.R. China.
authors.forename: Jianzhang
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Otolaryngology, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, P.R. China.
authors.forename: Jun
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Otolaryngology, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, P.R. China.
authors.forename: Changping
authors.initials: C
authors.lastname: Cai
authors.affiliation: Department of Otolaryngology, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, P.R. China.
authors.forename: Shili
authors.initials: S
authors.lastname: Wang
authors.affiliation: Department of Nuclear Medicine, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, P.R. China.
authors.forename: Shuai
authors.initials: S
authors.lastname: Liu
authors.affiliation: Department of Nuclear Medicine, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, P.R. China.
authors.forename: Shuo
authors.initials: S
authors.lastname: Shi
authors.affiliation: Department of Nuclear Medicine, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, P.R. China.
authors.forename: Yifan
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Nuclear Medicine, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, P.R. China.
authors.forename: Biao
authors.initials: B
authors.lastname: Li
keywords: baculovirus; coexpression; dual-gene delivery; dual-promoter; gene delivery vector