title: Lentiviral Vectors Mediate Long-Term and High Efficiency Transgene Expression in HEK 293T cells.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: 1. School of Biotechnology, Southern Medical University, Guangzhou, Guangdong, China.
authors.forename: Yingying
authors.initials: Y
authors.lastname: Mao
authors.affiliation: 1. School of Biotechnology, Southern Medical University, Guangzhou, Guangdong, China.
authors.forename: Renhe
authors.initials: R
authors.lastname: Yan
authors.affiliation: 2. Department of Biomedical Engineering, The Johns Hopkins University School of Medicine, Baltimore, USA.
authors.forename: Andrew
authors.initials: A
authors.lastname: Li
authors.affiliation: 1. School of Biotechnology, Southern Medical University, Guangzhou, Guangdong, China.
authors.forename: Yanling
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: 1. School of Biotechnology, Southern Medical University, Guangzhou, Guangdong, China.
authors.forename: Jinlong
authors.initials: J
authors.lastname: Li
authors.affiliation: 1. School of Biotechnology, Southern Medical University, Guangzhou, Guangdong, China.
authors.forename: Hongyan
authors.initials: H
authors.lastname: Du
authors.affiliation: 1. School of Biotechnology, Southern Medical University, Guangzhou, Guangdong, China.
authors.forename: Baihong
authors.initials: B
authors.lastname: Chen
authors.affiliation: 3. Beijing Minhai Biotechnology CO., LTD, Beijing, China.
authors.forename: Wenjin
authors.initials: W
authors.lastname: Wei
authors.affiliation: 4. Department of Pharmacology, University of Florida, Gainesville, Florida, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: 5. Department of Physiology and Functional Genomics, University of Florida, Gainesville, Florida, USA.
authors.forename: Colin
authors.initials: C
authors.lastname: Sumners
authors.affiliation: 3. Beijing Minhai Biotechnology CO., LTD, Beijing, China.
authors.forename: Haifa
authors.initials: H
authors.lastname: Zheng
authors.affiliation: 1. School of Biotechnology, Southern Medical University, Guangzhou, Guangdong, China.
authors.forename: Hongwei
authors.initials: H
authors.lastname: Li
mesh_terms: D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D011401:Promoter Regions, Genetic; D014161:Transduction, Genetic; D019076:Transgenes; D014759:Viral Envelope Proteins / Q000235:genetics; D055544:beta-Globins / Q000235:genetics
chemical_list: C087035:E1 protein, Hepatitis C virus; D014759:Viral Envelope Proteins; D055544:beta-Globins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: HCV E1.; HEK 293 cells; UCOE; insulator; lentiviral vector; promoters; protein production