title: Sleeping Beauty transposon-based system for rapid generation of HBV-replicating stable cell lines.
abstract: The stable HBV-replicating cell lines, which carry replication-competent HBV genome stably integrated into the genome of host cell, are widely used to evaluate the effects of antiviral agents. However, current methods to generate HBV-replicating cell lines, which are mostly dependent on random integration of foreign DNA via plasmid transfection, are less-efficient and time-consuming. To address this issue, we constructed an all-in-one Sleeping Beauty transposon system (denoted pTSMP-HBV vector) for robust generation of stable cell lines carrying replication-competent HBV genome of different genotype. This vector contains a Sleeping Beauty transposon containing HBV 1.3-copy genome with an expression cassette of the SV40 promoter driving red fluorescent protein (mCherry) and self-cleaving P2A peptide linked puromycin resistance gene (PuroR). In addition, a PGK promoter-driven SB100X hyperactive transposase cassette is placed in the outside of the transposon in the same plasmid.The HBV-replicating stable cells could be obtained from pTSMP-HBV transfected HepG2 cells by red fluorescence-activated cell sorting and puromycin resistant cell selection within 4-week. Using this system, we successfully constructed four cell lines carrying replication-competent HBV genome of genotypes A-D. The replication and viral protein expression profiles of these cells were systematically characterized. In conclusion, our study provides a high-efficiency strategy to generate HBV-replicating stable cell lines, which may facilitate HBV-related virological study.
authors.affiliation: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Public Health, Xiamen University, Xiamen 361102, China; National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, Xiamen University, Xiamen 361102, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Public Health, Xiamen University, Xiamen 361102, China; School of Life Science, Xiamen University, Xiamen 361102, China.
authors.forename: Tian-Ying
authors.initials: TY
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Public Health, Xiamen University, Xiamen 361102, China; National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, Xiamen University, Xiamen 361102, China.
authors.forename: Lin-Lin
authors.initials: LL
authors.lastname: Fang
authors.affiliation: Affiliated Quanzhou First Hospital by Fujian Medical University, Fujian, China.
authors.forename: Zi-Xuan
authors.initials: ZX
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Public Health, Xiamen University, Xiamen 361102, China; School of Life Science, Xiamen University, Xiamen 361102, China.
authors.forename: Liu-Wei
authors.initials: LW
authors.lastname: Song
authors.affiliation: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Public Health, Xiamen University, Xiamen 361102, China; School of Life Science, Xiamen University, Xiamen 361102, China.
authors.forename: Jia-Li
authors.initials: JL
authors.lastname: Cao
authors.affiliation: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Public Health, Xiamen University, Xiamen 361102, China; National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, Xiamen University, Xiamen 361102, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Yang
authors.affiliation: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Public Health, Xiamen University, Xiamen 361102, China; National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, Xiamen University, Xiamen 361102, China. Electronic address: yuanquan@xmu.edu.cn.
authors.forename: Quan
authors.initials: Q
authors.lastname: Yuan
authors.affiliation: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Public Health, Xiamen University, Xiamen 361102, China; National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, Xiamen University, Xiamen 361102, China.
authors.forename: Ning-Shao
authors.initials: NS
authors.lastname: Xia
mesh_terms: D046508:Culture Techniques; D004261:DNA Replication; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006367:HeLa Cells; D056945:Hep G2 Cells; D006515:Hepatitis B virus / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008164:Luminescent Proteins; D019895:Transposases / Q000235:genetics*; D014779:Virus Replication; D000097573:Red Fluorescent Protein
chemical_list: D008164:Luminescent Proteins; D019895:Transposases; D000097573:Red Fluorescent Protein; C494471:sleeping beauty transposase, human
keywords: Hepatitis B virus; Sleeping Beauty transposon; Stable cell line