Scherer Olga, Maeß Marten B, Lindner Saskia, Garscha Ulrike, Weinigel Christina, Rummler Silke, Werz Oliver, Lorkowski Stefan
Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich Schiller University Jena, Philosophenweg 14, 07743 Jena, Germany.
Nutritional Biochemistry and Physiology, Institute of Nutrition, Friedrich Schiller University Jena, Dornburger Str. 25, 07743 Jena, Germany.
J Immunol Methods. 2015 Jul;422:118-24. doi: 10.1016/j.jim.2015.04.007. Epub 2015 Apr 17.
Monocytes are an important constituent of the innate immune system. Therefore, manipulating gene expression of primary human monocytes is a crucial mean to study and characterize the functions of targeted proteins in monocytes. Gene silencing by transfection of cells with small interfering RNA (siRNA) leading to the degradation of the corresponding mRNA and thus to reduced target protein levels is an important tool to investigate gene and protein function of interest. However, non-viral transfection of primary monocytes is challenging because siRNA uptake by these suspended cells is tricky, and the individual cells vary among different donors and do not proliferate. Here, we describe a procedure for efficient non-viral transfection of primary human monocytes isolated from peripheral blood, which maintains cell viability and cell functions, such as responsiveness to stimuli like LPS and IL-10. Nucleofection was used as an electroporation technique that enables efficient introduction of siRNA and silencing of target genes. Using a modification of our previously published protocol for the fast-proliferating THP-1 monocytic cell line, we transfected primary human monocytes with siRNA targeting 5-lipoxygenase (5-LO). In fact, we successfully downregulated 5-LO mRNA resulting in reduced protein levels and enzymatic activity.
单核细胞是先天性免疫系统的重要组成部分。因此，操纵原代人单核细胞的基因表达是研究和表征单核细胞中靶向蛋白功能的关键手段。通过用小干扰RNA（siRNA）转染细胞导致相应mRNA降解从而降低靶蛋白水平来实现基因沉默，是研究感兴趣的基因和蛋白功能的重要工具。然而，原代单核细胞的非病毒转染具有挑战性，因为这些悬浮细胞摄取siRNA很棘手，而且不同供体的单个细胞存在差异且不会增殖。在此，我们描述了一种从外周血中分离的原代人单核细胞的高效非病毒转染方法，该方法可维持细胞活力和细胞功能，如对LPS和IL-10等刺激的反应性。核转染被用作一种电穿孔技术，可有效导入siRNA并沉默靶基因。通过对我们之前发表的针对快速增殖的THP-1单核细胞系的方案进行修改，我们用靶向5-脂氧合酶（5-LO）的siRNA转染原代人单核细胞。事实上，我们成功下调了5-LO mRNA，导致蛋白水平和酶活性降低。
