Lentiviral Vectors Mediate Long-Term and High Efficiency Transgene Expression in HEK 293T cells

Abstract

Objectives: Lentiviral vectors have been used successfully to rapidly produce decigram quantities of active recombinant proteins in mammalian cell lines. To optimize the protein production platform, the roles of Ubiquitous Chromatin Opening Element (UCOE), an insulator, and selected promoters were evaluated based on efficiency and stability of foreign gene expression mediated by lentiviral vectors.

Methods: Five lentiviral vectors, pFIN-EF1α-GFP-2A-mCherH-WPRE containing EF1α promoter and HS4 insulator, p'HR.cppt.3'1.2kb-UCOE-SFFV-eGFP containing SFFV promoter and UCOE, pTYF-CMV(β-globin intron)-eGFP containing CMV promoter and β-globin intron, pTYF-CMV-eGFP containing CMV promoter, and pTYF-EF1α-eGFP with EF1α promoter were packaged, titered, and then transduced into 293T cells (1000 viral genomes per cell). The transduced cells were passaged once every three days at a ratio of 1:10. Expression level and stability of the foreign gene, green fluorescence protein (GFP), was evaluated using fluorescent microscopy and flow cytometry. Furthermore, we constructed a hepatitis C virus (HCV) E1 recombinant lentiviral vector, pLV-CMV-E1, driven by the CMV promoter. This vector was packaged and transduced into 293T cells, and the recombinant cell lines with stable expression of E1 protein were established by limiting dilution.

Results: GFP expression in 293T cells transduced with the five lentiviral vectors peaked between passages 3 and 5 and persisted for more than 5 weeks. The expression was prolonged in the cells transduced with TYF-CMV (β-globin intron)-eGFP or TYF-CMV-eGFP, demonstrating less than a 50% decrease even at 9 weeks post transduction (p>0.05). The TYF-CMV-eGFP-transduced cells began with a higher level of GFP expression than other vectors did. The percentage of GFP positive cells for any of the five lentiviral vectors sustained over time. Moreover, the survival rates of all transfected cells exceeded 80% at both 5 and 9 weeks post transduction. Surprisingly, neither the HS4 insulator nor the UCOE sequence improved the GFP expression level or stability. Clonal cell lines with HCV E1 gene were generated from LV-CMV-E1 vector-infected 293T cells. A representative recombinant cell line maintained stable E1expression for at least 9 weeks without significant difference in morphology compared with untreated 293T cells.

Conclusion: The results suggest that all five vectors can stably transduce 293T cells, producing long term transgene expression with different efficiencies. However, neither the insulator nor the UCOE improved the GFP expression. The vectors containing the promoter CMV or CMV (β-globin intron) generated the highest gene expressions, manifesting as more favorable candidates for recombinant protein production in HEK293T cells.

Keywords: HCV E1.; HEK 293 cells; UCOE; insulator; lentiviral vector; promoters; protein production.

Figure 1

Schemes of the lentiviral transfer vectors used in the study. (A) pFIN-EF1α-GFP-2A-mCherH-WPRE containing EF1α promoter and HS4 insulator, (B) p'HR.cppt.3'1.2kb-UCOE-SFFV-eGFP containing SFFV promoter and UCOE, (C) pTYF-CMV(β-globin intron)-eGFP containing CMV promoter and β-globin intron, (D) pTYF-CMV-eGFP containing CMV promoter, and (E) pTYF-EF1α-eGFP with EF1α promoter. Abbreviations: EF1 α - elongation factor 1 α promoter; SFFV, spleen focus forming virus (SFFV) promoter; GFP - green fluorescent protein; mCher - cherry fluorescent protein; 2A - porcine teschovirus (pTV1) 2A-like cleavage peptide; bGH-bovine growth hormone derived polyadenylation signal.

Figure 2

Lentiviral-mediated transduction of GFP into HEK 293T cells. HEK 293T cells were transduced with lentiviral vectors FIN-EF1α-GFP-2A-mCherH-WPRE (A), HR.cppt.3'1.2kb-UCOE-SFFV-eGFP (B) , TYF-CMV (β-globin intron)-eGFP (C), TYF-CMV-eGFP (D) or TYF-EF1α-eGFP (E) as described in the method. The images were captured by a fluorescent (upper panels) or bright light (lower panels) microscope at 1 week, 5 weeks and 9 weeks post transduction using same exposure time.

Figure 2

Lentiviral-mediated transduction of GFP into HEK 293T cells. HEK 293T cells were transduced with lentiviral vectors FIN-EF1α-GFP-2A-mCherH-WPRE (A), HR.cppt.3'1.2kb-UCOE-SFFV-eGFP (B) , TYF-CMV (β-globin intron)-eGFP (C), TYF-CMV-eGFP (D) or TYF-EF1α-eGFP (E) as described in the method. The images were captured by a fluorescent (upper panels) or bright light (lower panels) microscope at 1 week, 5 weeks and 9 weeks post transduction using same exposure time.

Figure 2

Lentiviral-mediated transduction of GFP into HEK 293T cells. HEK 293T cells were transduced with lentiviral vectors FIN-EF1α-GFP-2A-mCherH-WPRE (A), HR.cppt.3'1.2kb-UCOE-SFFV-eGFP (B) , TYF-CMV (β-globin intron)-eGFP (C), TYF-CMV-eGFP (D) or TYF-EF1α-eGFP (E) as described in the method. The images were captured by a fluorescent (upper panels) or bright light (lower panels) microscope at 1 week, 5 weeks and 9 weeks post transduction using same exposure time.

Figure 2

Lentiviral-mediated transduction of GFP into HEK 293T cells. HEK 293T cells were transduced with lentiviral vectors FIN-EF1α-GFP-2A-mCherH-WPRE (A), HR.cppt.3'1.2kb-UCOE-SFFV-eGFP (B) , TYF-CMV (β-globin intron)-eGFP (C), TYF-CMV-eGFP (D) or TYF-EF1α-eGFP (E) as described in the method. The images were captured by a fluorescent (upper panels) or bright light (lower panels) microscope at 1 week, 5 weeks and 9 weeks post transduction using same exposure time.

Figure 2

Lentiviral-mediated transduction of GFP into HEK 293T cells. HEK 293T cells were transduced with lentiviral vectors FIN-EF1α-GFP-2A-mCherH-WPRE (A), HR.cppt.3'1.2kb-UCOE-SFFV-eGFP (B) , TYF-CMV (β-globin intron)-eGFP (C), TYF-CMV-eGFP (D) or TYF-EF1α-eGFP (E) as described in the method. The images were captured by a fluorescent (upper panels) or bright light (lower panels) microscope at 1 week, 5 weeks and 9 weeks post transduction using same exposure time.

Figure 3

Comparison of different lentiviral vectors containing different elements on GFP positive efficiency and GFP MFI. Flow cytometry was performed at 5 weeks and 9 weeks after HEK 293T cells were transduced with lentiviral vectors. (A) GFP positive efficiency at 5 weeks post transduction. (B) The GFP MFI at 5 weeks and 9 weeks post transduction.

Figure 4

The sustaining expression of HCV E1 in clonal 293T cell lines established using the LV-CMV-E1 vector. Five microliters of medium, collected from a representative cell clone of HCV E1 during 9 weeks, were separated on SDS-PAGE and analyzed by western blotting with an anti-HCV polyclonal antibody. (A) A representative western blot showing sustaining expressing of HCV E1 from 1 week to 9 weeks. (B) Bar graphs showing the expression of HCV E1 levels in each time point. Data are means ±S.E.M. from 3 experiments. Y-axis is HCV E1 protein levels expressed as relative optical density values in arbitrary units. eGFP: the 293T cell transduced by LV-CMV-eGFP; 293T: untreated 293T cells.