Jantsch Jonathan, Turza Nadine, Volke Melanie, Eckardt Kai-Uwe, Hensel Michael, Steinkasserer Alexander, Willam Carsten, Prechtel Alexander T
Department of Nephrology and Hypertension, University Hospital Erlangen, Friedrich-Alexander University of Erlangen-Nuremberg, Krankenhausstrasse 12, Erlangen, Germany.
J Immunol Methods. 2008 Aug 20;337(1):71-7. doi: 10.1016/j.jim.2008.04.004. Epub 2008 Apr 28.
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.
RNA干扰（RNAi）介导的选择性基因沉默已被证明是一种有效靶向调控细胞功能的方法。在本研究中，我们首次描述了电穿孔作为一种将小干扰RNA（siRNA）导入小鼠骨髓来源树突状细胞（BM-DC）的合适且有效的方法。使用荧光素标记的非沉默siRNA双链体，我们建立了一种电穿孔方案，常规可产生>90%的阳性细胞。我们研究了siRNA电穿孔对BM-DC活力、表型以及诱导同种异体T细胞增殖能力的影响。最后，使用针对MAPK1和转录因子HIF-1α的siRNA，我们能够证明在电穿孔的BM-DC中细胞mRNA和蛋白质水平的有效敲低。此外，敲低转录因子HIF-1α会阻碍HIF-1α靶基因的低氧诱导。因此，我们提出将siRNA电穿孔导入小鼠BM-DC是一种无需使用化学转染试剂即可调控BM-DC功能的有效方法。就基于脂质的转染试剂对BM-DC免疫生物学的有害影响而言，这种新方法优于脂质转染法。
