1] CNRS, Institut de Pharmacologie et de Biologie Structurale, Toulouse, France [2] Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, Université Paul Sabatier, Toulouse, France.
Division of Hematology & Oncology, Tisch Cancer Institute, Icahn School of Medicine at Mt Sinai, New York, NY, USA.
Immunol Cell Biol. 2014 Sep;92(8):699-708. doi: 10.1038/icb.2014.39. Epub 2014 Jun 3.
Mononuclear phagocytes (MP) comprise monocytes, macrophages (MΦ) and dendritic cells (DC), including their lineage-committed progenitors, which together have an eminent role in health and disease. Lipid-based siRNA-mediated gene inactivation is an established approach to investigate gene function in MP cells. However, although there are few protocols dedicated for siRNA-mediated gene inactivation in primary human DC and MΦ, there are none available for primary human monocytes. Moreover, there is no available method to perform comparative studies of a siRNA-mediated gene silencing in primary monocytes and other MP cells. Here, we describe a protocol optimized for the lipid-based delivery of siRNA to perform gene silencing in primary human blood monocytes, which is applicable to DCs, and differs from the classical route of siRNA delivery into MΦs. Along with this protocol, we provide a comparative analysis of how monocytes, DC and MΦ are efficiently transfected with the target siRNA without affecting cell viability, resulting in strong gene knockdown efficiency, including the simultaneous inactivation of two genes. Moreover, siRNA delivery does not affect classical functions in MP such as differentiation, phagocytosis and migration, demonstrating that this protocol does not induce non-specific major alterations in these cells. As a proof-of-principle, a functional analysis of hematopoietic cell kinase (Hck) shows for the first time that this kinase regulates the protease-dependent migration mode in human monocytes. Collectively, this protocol enables efficient gene inactivation in primary MP, suggesting a wide spectrum of applications such as siRNA-based high-throughput screening, which could ultimately improve our knowledge about MP biology.
单核吞噬细胞（MP）包括单核细胞、巨噬细胞（MΦ）和树突状细胞（DC），包括其谱系定向祖细胞，它们在健康和疾病中具有重要作用。基于脂质的 siRNA 介导的基因失活是研究 MP 细胞中基因功能的一种既定方法。然而，尽管有一些专门用于原代人 DC 和 MΦ中 siRNA 介导的基因失活的方案，但没有适用于原代人单核细胞的方案。此外，没有可用的方法来对原代单核细胞和其他 MP 细胞中的 siRNA 介导的基因沉默进行比较研究。在这里，我们描述了一种优化的基于脂质的 siRNA 递送来实现原代人血液单核细胞中基因沉默的方案，该方案适用于 DC，并且与经典的 MΦ 中 siRNA 递送途径不同。随着这个方案，我们提供了一个比较分析，说明单核细胞、DC 和 MΦ 如何在不影响细胞活力的情况下有效地转染靶 siRNA，从而产生强烈的基因敲低效率，包括同时失活两个基因。此外，siRNA 递送不会影响 MP 中的经典功能，如分化、吞噬和迁移，表明该方案不会在这些细胞中引起非特异性的主要改变。作为原理验证，造血细胞激酶（Hck）的功能分析首次表明，这种激酶调节人单核细胞中依赖蛋白酶的迁移模式。总的来说，这个方案能够在原代 MP 中有效地进行基因失活，这表明它有广泛的应用，如基于 siRNA 的高通量筛选，这最终可以提高我们对 MP 生物学的认识。
