title: Highly reproducible transient transfections for the study of hepatitis B virus replication based on an internal GFP reporter system.
abstract: High throughput studies of hepatitis B virus (HBV) replication are limited by the absence of convenient and reliable in vitro systems that can be utilised to examine mutations emerging as a consequence of selective pressures such as drug therapy, immune response and genotypic evolution. We have developed an efficient and reproducible method of transfecting a human hepatoma cell line (Huh7) with a replication competent clone of HBV, which utilises an internal reporter of transfection efficiency. Wild type (WT) and a common lamivudine (LMV) resistant mutant virus characterised by a methionine to isoleucine mutation in the highly conserved YMDD motif of the polymerase gene were used to highlight the utility of this in vitro transfection procedure in studying common HBV mutations. The presence of an internal GFP reporter increased the efficiency of generating highly reproducible results and facilitated multiple forms of analysis from a single transfection accurately.
authors.affiliation: Department of Medicine (RMH), University of Melbourne, Vic., Australia.
authors.forename: Theo
authors.initials: T
authors.lastname: Gouskos
authors.forename: Fiona
authors.initials: F
authors.lastname: Wightman
authors.forename: Sharon R
authors.initials: SR
authors.lastname: Lewin
authors.forename: Joseph
authors.initials: J
authors.lastname: Torresi
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology; D015152:Blotting, Northern; D045744:Cell Line, Tumor; D004279:DNA, Viral / Q000032:analysis; D024882:Drug Resistance, Viral; D005434:Flow Cytometry; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D006515:Hepatitis B virus / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D006801:Humans; D019259:Lamivudine / Q000494:pharmacology; D009154:Mutation; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D012367:RNA, Viral / Q000032:analysis; D015203:Reproducibility of Results; D014162:Transfection; D014779:Virus Replication
chemical_list: D000998:Antiviral Agents; D004279:DNA, Viral; D012333:RNA, Messenger; D012367:RNA, Viral; D049452:Green Fluorescent Proteins; D019259:Lamivudine