Sleeping Beauty transposon-based system for rapid generation of HBV-replicating stable cell lines

J Virol Methods. 2016 Aug;234:96-100. doi: 10.1016/j.jviromet.2016.04.010. Epub 2016 Apr 22.

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.

Keywords: Hepatitis B virus; Sleeping Beauty transposon; Stable cell line.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Culture Techniques
  • DNA Replication
  • Flow Cytometry
  • Gene Transfer Techniques
  • Genetic Vectors
  • HeLa Cells
  • Hep G2 Cells
  • Hepatitis B virus / genetics
  • Hepatitis B virus / physiology*
  • Humans
  • Luminescent Proteins
  • Transposases / genetics*
  • Virus Replication*

Substances

  • Luminescent Proteins
  • red fluorescent protein
  • Transposases
  • sleeping beauty transposase, human