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Efficient genetic modification of murine dendritic cells by electroporation with mRNA

Cancer Gene Therapy volume 9, pages 787–797 (2002)Cite this article

Abstract

Recently, human dendritic cells (DCs) pulsed with mRNA encoding a broad range of tumor antigens have proven to be potent activators of a primary anti–tumor-specific T-cell response in vitro. The aim of this study was to improve the mRNA pulsing of murine DC. Compared to a standard lipofection protocol and passive pulsing, electroporation was, in our hands, the most efficient method. The optimal conditions to electroporate murine bone marrow–derived DCs with mRNA were determined using enhanced green fluorescent protein and a truncated form of the nerve growth factor receptor. We could obtain high transfection efficiencies around 70–80% with a mean fluorescence intensity of 100–200. A maximal expression level was reached 3 hours after electroporation. A clear dose–response effect was seen depending on the amount of mRNA used. Importantly, the electroporation process did not affect the viability nor the allostimulatory capacity or phenotype of the DC. To study the capacity of mRNA-electroporated DCs to present antigen in the context of MHC classes I and II, we made use of chimeric constructs of ovalbumin. The dose-dependent response effect and the duration of presentation were also determined. Together, these results demonstrate that mRNA electroporation is a useful method to generate genetically modified murine DC, which can be used for preclinical studies testing immunotherapeutic approaches.

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Acknowledgements

We thank Muriel Moser for helpful discussion, Hilde Revets for kindly providing the 25-D1.16 hybridoma cells, Elsy Vaeremans, Peggy Verbuyst, and Gert De Block for technical assistance, and Yasmina Essam for secretarial assistance. This work was supported by grants to KT from the Fund for Scientific Research-Flanders (FWO-Vlaanderen), the Institute for Science and Technology (IWT), the Ministry of Science (IUAP/PAI IV), De Algemene Spaar-en Lijfrentekas (ASLK/CGER), and De Belgische Federatie voor Kankerbestrijding.

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  1. Laboratory of Physiology-Immunology of the Medical School of the Vrije Universiteit Brussel (VUB), Brussels, Belgium

    Sonja Van Meirvenne, Lieven Straetman, Carlo Heirman, Melissa Dullaers, Catherine De Greef & Kris Thielemans

  2. Laboratory of Experimental Hematology, University of Antwerp (UIA), Antwerp University Hospital (UZA), Edegem, Belgium

    Viggo Van Tendeloo

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Van Meirvenne, S., Straetman, L., Heirman, C. et al. Efficient genetic modification of murine dendritic cells by electroporation with mRNA. Cancer Gene Ther 9, 787–797 (2002). https://doi.org/10.1038/sj.cgt.7700499

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