Efficient genetic modification of murine dendritic cells by electroporation with mRNA.
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.
Laboratory of Physiology-Immunology of the Medical School of the Vrije Universiteit Brussel (VUB), Brussels, Belgium.
Sonja
S
Van Meirvenne
Lieven
L
Straetman
Carlo
C
Heirman
Melissa
M
Dullaers
Catherine
C
De Greef
Viggo
V
Van Tendeloo
Kris
K
Thielemans
D000818:Animals; D017951:Antigen Presentation / Q000276:immunology*; D002455:Cell Division; D002645:Chickens; D017931:DNA Primers / Q000737:chemistry; D003713:Dendritic Cells / Q000276:immunology*; D018274:Electroporation / Q000379:methods*; D005260:Female; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D005805:Genes, MHC Class I / Q000235:genetics; D005802:Genes, MHC Class II / Q000235:genetics; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008213:Lymphocyte Activation; D008223:Lymphoma / Q000276:immunology / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D051379:Mice; D008810:Mice, Inbred C57BL; D010047:Ovalbumin / Q000378:metabolism; D010446:Peptide Fragments / Q000276:immunology; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics*; D020800:Receptor, Nerve Growth Factor / Q000235:genetics / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology; D014158:Transcription, Genetic; D025801:Ubiquitin / Q000494:pharmacology
D017931:DNA Primers; D008164:Luminescent Proteins; D010446:Peptide Fragments; D012333:RNA, Messenger; D020800:Receptor, Nerve Growth Factor; D025801:Ubiquitin; D049452:Green Fluorescent Proteins; D010047:Ovalbumin