Abstract
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 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 in vitro and in vivo.
Highlights
Gene therapy has been defined as the deliberate introduction of genetic material into cells in order to treat or prevent a disease
Following the construction of the pFastBac Dual‐CMV‐enhanced green fluorescent protein (EGFP)‐CMV‐glial cell line‐derived neurotrophic factor (GDNF) plasmid, the size was evaluated by 1.2% agarose gel electrophoresis
The PH and p10 promoters in the pFastBac Dual vector were replaced by CMV promoters, whose directions were consistent with the initial direction of the PH or p10 promoter
Summary
Gene therapy has been defined as the deliberate introduction of genetic material into cells in order to treat or prevent a disease. A number of studies have focused on creating more efficient, stable and safe gene therapy vectors. Viral and non‐viral vectors are the two main groups of vectors that are used in gene therapy. Used viral vectors include adenovirus, adeno‐associated virus, retrovirus and lentivirus. These vectors possess limitations, including possible safety problems [1,2] and challenges for large scale production. In contrast to the commonly employed viral vectors, baculovirus has unique properties of inherent safety, ease and speed of virus generation in high quantities, low cytotoxicity and extreme transgene capacity [3]. The baculovirus vector is being increasingly applied and continually developed for efficient gene therapy
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