title: mRNA-based electrotransfection of human dendritic cells and induction of cytotoxic T lymphocyte responses against the telomerase catalytic subunit (hTERT).
abstract: Dendritic cells (DCs) are recognised as the most potent antigen-presenting cells for induction of cellular immune responses, and vaccination with DCs pulsed with antigens has emerged as a promising strategy for generating protective immunity in mammals. We have developed a transfection method that uses in vitro synthesised mRNA and square-wave electroporation for transient expression in DCs and other cell types. The method is highly efficient and produces almost complete transfection of cells in culture. When using mRNA encoding the enhanced green fluorescence protein (EGFP), highest expression in DCs occurred on the second day after transfection and produced a 76-fold increase in mean fluorescence above background. High levels of expression were maintained for at least 5 days post-transfection. In comparison, square-wave electroporation of DCs with EGFP plasmid DNA yielded 15% transfected cells and a 28-fold increase of mean fluorescence. DCs transfected with mRNA encoding the telomerase catalytic subunit (hTERT) acquired strong telomerase activity and were capable of eliciting a hTERT-specific cytotoxic T lymphocyte (CTL) response in vitro.
authors.affiliation: Section for Immunotherapy, Department of Immunology, The Norwegian Radium Hospital, Cancer Research Institute, University of Oslo, N-0310 Montebello, Oslo, Norway. stein.saboe-larssen@samfunnsmed.uio.no
authors.forename: Stein
authors.initials: S
authors.lastname: Saebøe-Larssen
authors.forename: Ellen
authors.initials: E
authors.lastname: Fossberg
authors.forename: Gustav
authors.initials: G
authors.lastname: Gaudernack
mesh_terms: D017951:Antigen Presentation / Q000235:genetics* / Q000276:immunology*; D020134:Catalytic Domain / Q000235:genetics / Q000276:immunology; D002478:Cells, Cultured; D003602:Cytotoxicity, Immunologic; D004268:DNA-Binding Proteins; D003713:Dendritic Cells / Q000276:immunology*; D018274:Electroporation; D006801:Humans; D012333:RNA, Messenger / Q000235:genetics / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D019098:Telomerase / Q000235:genetics* / Q000276:immunology*; D014162:Transfection / Q000379:methods*
chemical_list: D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D019098:Telomerase