Small interfering RNA (siRNA) delivery into murine bone marrow-derived macrophages by electroporation.
Selective gene silencing by RNA interference (RNAi) is a valuable tool for the targeted manipulation of the development and/or function of cells. Using a fluorescein-labeled non-silencing siRNA duplex, we established a protocol for the electroporation of primary mouse macrophages which routinely yielded >95% transfected cells. Electroporation of siRNAs directed against MAPK1 and CD86 led to an efficient knock-down of cellular protein in bone marrow-derived mouse macrophages (BM-Mphi). Importantly, the electroporation procedure did not impair the viability of BM-Mphi, their ability to ingest or degrade E. coli or their capacity to express iNOS mRNA, to produce NO or to upregulate TNF and IL-6 mRNA in response to inflammatory stimuli such as LPS. Therefore, we propose that electroporation of silencing siRNAs into murine BM-Mphi is a highly efficient method to manipulate gene expression of BM-Mphi that does not cause toxicity or a non-specific alteration of macrophage biology.
Wiese
Melanie
M
Microbiology Institute-Clinical Microbiology, Immunology and Hygiene, University Clinic Erlangen, Friedrich-Alexander University of Erlangen-Nuremberg, Erlangen, Germany.
Castiglione
Kirstin
K
Hensel
Michael
M
Schleicher
Ulrike
U
Bogdan
Christian
C
Jantsch
Jonathan
J
D000818:Animals; D051940:B7-2 Antigen / Q000235:genetics; D002470:Cell Survival; D002478:Cells, Cultured; D018274:Electroporation; D005260:Female; D015850:Interleukin-6 / Q000235:genetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology / Q000276:immunology / Q000378:metabolism* / Q000382:microbiology; D051379:Mice; D008810:Mice, Inbred C57BL; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics; D052247:Nitric Oxide Synthase Type II / Q000235:genetics; D010587:Phagocytosis; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
D051940:B7-2 Antigen; C497965:Cd86 protein, mouse; D015850:Interleukin-6; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; C535148:Mapk1 protein, mouse; D019950:Mitogen-Activated Protein Kinase 1