Ž . Journal of Immunological Methods 259 2002 191–203 www.elsevier.comrlocaterjim Recombinant Technology mRNA-based electrotransfection of human dendritic cells and induction of cytotoxic T lymphocyte responses against the ž / telomerase catalytic subunit hTERT Stein Sæbøe-Larssen ) , Ellen Fossberg, Gustav Gaudernack Section for Immunotherapy, Department of Immunology, The Norwegian Radium Hospital, Cancer Research Institute, UniÕersity of Oslo, N-0310 Montebello, Oslo, Norway Received 10 June 2001; received in revised form 2 September 2001; accepted 2 September 2001 Abstract Ž . 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 Ž . 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 Ž . increase of mean fluorescence. DCs transfected with mRNA encoding the telomerase catalytic subunit hTERT acquired Ž . strong telomerase activity and were capable of eliciting a hTERT-specific cytotoxic T lymphocyte CTL response in vitro. q 2002 Elsevier Science B.V. All rights reserved. Keywords: Dendritic cells; T lymphocytes; Telomerase; mRNA transfection; Electroporation 1. Introduction The last two decades have seen intensive efforts to clone and characterise genes from pathogenic AbbreÕiations: BG, background level; CDS, coding sequence; CTL, cytotoxic T lymphocyte; DC, dendritic cell; MCS, multiple cloning site; ORF, open reading frame; PBMC, peripheral blood mononuclear cell; TRAP, telomeric repeat amplification protocol. ) Corresponding author. Tel.: q 47-22934754; fax: q 47- 22934836. E-mail address: stein.saboe-larssen@samfunnsmed.uio.no Ž . S. Sæbøe-Larssen . sources and tumour tissues. Based on this knowl- edge, new and important approaches for medical treatment and vaccination regimes have emerged us- ing such genes and mutations as antigens to induce protective immune responses. In nearly all cases, DNA has been used for transfection purposes be- cause of its inherent stability and its ability to inte- grate into the host genome to produce stable trans- fectants. Because genetic vaccines are relatively inexpensive and easy to manufacture, and can be administered directly by injection into skin and other tissues, their immunogenicity and efficacy have been 0022-1759r02r$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. Ž . PII: S0022-1759 01 00506-3