Small interfering RNA (siRNA) delivery into murine bone marrow-derived dendritic cells by electroporation.
Selective gene silencing by RNA interference (RNAi) has been shown to be an efficient method for the targeted manipulation of cellular functions. In this study we describe for the first time electroporation as a suitable and efficient method for the delivery of small interfering RNA (siRNA) into murine bone marrow-derived dendritic cells (BM-DC). Using a fluorescein-labeled non-silencing siRNA duplex, we established an electroporation protocol yielding routinely >90% positive cells. We investigated the effects of siRNA electroporation on BM-DC viability, phenotype and ability to induce allogeneic T cell proliferation. Finally, using siRNAs directed against MAPK1 and the transcription factor HIF-1alpha we were able to demonstrate an efficient knock down of cellular mRNA- and protein level in electroporated BM-DC. Furthermore, knocking down the transcription factor HIF-1alpha impeded hypoxic induction of HIF-1alpha target genes. We therefore propose siRNA electroporation into murine BM-DC as an efficient method to manipulate BM-DC function without the use of chemical transfection reagents. This new approach is superior to lipofection regarding detrimental effects of lipid-based transfection agents on BM-DC immunobiology.
Department of Nephrology and Hypertension, University Hospital Erlangen, Friedrich-Alexander University of Erlangen-Nuremberg, Krankenhausstrasse 12, Erlangen, Germany. jonathan.jantsch@uk-erlangen.de
Jonathan
J
Jantsch
Nadine
N
Turza
Melanie
M
Volke
Kai-Uwe
KU
Eckardt
Michael
M
Hensel
Alexander
A
Steinkasserer
Carsten
C
Willam
Alexander T
AT
Prechtel
D000818:Animals; D001854:Bone Marrow Cells / Q000201:enzymology / Q000276:immunology / Q000378:metabolism*; D015687:Cell Hypoxia; D002470:Cell Survival; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000201:enzymology / Q000276:immunology / Q000378:metabolism*; D018274:Electroporation; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism; D008055:Lipids; D008213:Lymphocyte Activation; D051379:Mice; D008810:Mice, Inbred C57BL; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism*; D013601:T-Lymphocytes / Q000276:immunology; D014162:Transfection / Q000379:methods*
C497443:Hif1a protein, mouse; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008055:Lipids; C086724:Lipofectamine; D034741:RNA, Small Interfering; D019950:Mitogen-Activated Protein Kinase 1