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 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.

Keywords: baculovirus; coexpression; dual-gene delivery; dual-promoter; gene delivery vector.

Figure 1

Map of the pFastBac Dual vector.

Figure 2

Agarose gel electrophoresis of the recombinant plasmid and bacmid DNA PCR products. Lane 1, pFastBac Dual-CMV-EGFP-CMV-GDNF plasmid; lane 2, PCR product of recombinant bacmid DNA amplified with pUC/M13 forward and reverse primers; lane 3, PCR product of recombinant bacmid DNA amplified with the specific and pUC/M13 reverse primers; lane 4, PCR product of non-recombinant bacmid DNA amplified with pUC/M13 forward and reverse primers; lane M1, DL 15,000 DNA marker; lane M2, DL 1,000 DNA marker; PCR, polymerase chain reaction; CMV, cytomegalovirus; EGFP, enhanced green fluorescent protein; GDNF, glial cell line-derived neurotrophic factor.

Figure 3

Confirmation of EGFP and GDNF coexpression in transfected HEK 293T cells and transduced HeLa cells. (A) Green fluorescence from EGFP and (B) red fluorescence immunostained by anti-GDNF antibody in HEK 293T cells transfected with the recombinant plasmid and Lipofectamine 2000. (C) Green fluorescence from EGFP and (D) red fluorescence immunostained by anti-GDNF antibody in HeLa cells transduced by the recombinant baculovirus. EGFP, enhanced green fluorescent protein; GDNF, glial cell line-derived neurotrophic factor; HEK, human embryonic kidney.

Figure 4

Western blot analysis of GDNF expression in HeLa cells transduced with the recombinant baculovirus. Lane 1, infected cell lysate; lane 2, non-infected cell lysate; GDNF, glial cell line-derived neurotrophic factor.