Feasibility of using a dual‑promoter recombinant baculovirus vector to coexpress EGFP and GDNF in mammalian cells

Wang,Jianzhang; Wang,Jun; Cai,Changping; Wang,Shili; Liu,Shuai; Shi,Shuo; Zhang,Yifan; Li,Biao

doi:10.3892/etm.2014.1655

June-2014 Volume 7 Issue 6

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June-2014 Volume 7 Issue 6

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Article

Feasibility of using a dual‑promoter recombinant baculovirus vector to coexpress EGFP and GDNF in mammalian cells

Authors:
- Jianzhang Wang
- Jun Wang
- Changping Cai
- Shili Wang
- Shuai Liu
- Shuo Shi
- Yifan Zhang
- Biao Li
View Affiliations / Copyright

Affiliations: 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
Pages: 1549-1554
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Published online on: March 31, 2014

https://doi.org/10.3892/etm.2014.1655
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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.

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Journals

International Journal of Molecular Medicine

International Journal of Oncology

Molecular Medicine Reports

Oncology Reports

Experimental and Therapeutic Medicine

Oncology Letters

Biomedical Reports

Molecular and Clinical Oncology

World Academy of Sciences Journal

International Journal of Functional Nutrition

International Journal of Epigenetics

Medicine International

Feasibility of using a dual‑promoter recombinant baculovirus vector to coexpress EGFP and GDNF in mammalian cells

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