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.
Keywords: 5-Lipoxygenase; Electroporation; Monocytes; Nucleofection; Transfection; siRNA.
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