Microbiology Institute-Clinical Microbiology, Immunology and Hygiene, University Clinic Erlangen, Friedrich-Alexander University of Erlangen-Nuremberg, Erlangen, Germany.
J Immunol Methods. 2010 Feb 28;353(1-2):102-10. doi: 10.1016/j.jim.2009.12.002. Epub 2009 Dec 16.
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.
通过 RNA 干扰（RNAi）进行选择性基因沉默是一种有价值的工具，可用于靶向操纵细胞的发育和/或功能。使用荧光素标记的非沉默 siRNA 双链体，我们建立了一种用于原代小鼠巨噬细胞电穿孔的方案，该方案常规可获得>95％的转染细胞。针对 MAPK1 和 CD86 的 siRNA 的电穿孔导致骨髓来源的小鼠巨噬细胞（BM-Mphi）中细胞蛋白的有效敲低。重要的是，电穿孔过程不会损害 BM-Mphi 的活力，不会损害它们摄取或降解大肠杆菌的能力，也不会损害它们表达 iNOS mRNA 的能力，不会产生 NO，也不会在响应炎性刺激物（例如 LPS）时上调 TNF 和 IL-6 mRNA。因此，我们提出将沉默 siRNA 电穿孔到鼠 BM-Mphi 中是一种高效的方法，可以操纵 BM-Mphi 的基因表达，而不会引起毒性或巨噬细胞生物学的非特异性改变。
