Effective Knockdown of Gene Expression in Primary Microglia With siRNA and Magnetic Nanoparticles Without Cell Death or Inflammation.

Alejandro Carrillo-Jimenez,Mar Puigdellívol,Jose Luis Venero,Peter Stgeorge-Hyslop,Guy Charles Brown,Anna Vilalta,Miguel Angel Burguillos

doi:10.3389/fncel.2018.00313

Abstract

Microglia, the resident immune cells of the brain, have multiple functions in physiological and pathological conditions, including Alzheimer’s disease (AD). The use of primary microglial cell cultures has proved to be a valuable tool to study microglial biology under various conditions. However, more advanced transfection methodologies for primary cultured microglia are still needed, as current methodologies provide low transfection efficiency and induce cell death and/or inflammatory activation of the microglia. Here, we describe an easy, and effective method based on the Glial-Mag method (OZ Biosciences) using magnetic nanoparticles and a magnet to successfully transfect primary microglia cells with different small interfering RNAs (siRNAs). This method does not require specialist facilities or specific training and does not induce cell toxicity or inflammatory activation. We demonstrate that this protocol successfully decreases the expression of two key genes associated with AD, the triggering receptor expressed in myeloid cells 2 (TREM2) and CD33, in primary microglia cell cultures.

Highlights

Microglia are the resident tissue macrophages of the central nervous system (CNS) where they survey for insults that may affect the homeostasis of the system (Kettenmann et al, 2011; Boche et al, 2013)
We analyzed the efficacy of knockdown by siTREM2 and siCD33 at the mRNA level measured by RT-qPCR in mouse primary cortical microglia
We present a simple method for successfully deliver small interfering RNAs (siRNAs) and knockdown of gene expression in primary microglia based on binding of the siRNA to magnetic nanoparticles and magnet-induced penetration of the cells

Summary

Introduction

Microglia are the resident tissue macrophages of the central nervous system (CNS) where they survey for insults that may affect the homeostasis of the system (Kettenmann et al, 2011; Boche et al, 2013) They play several roles under physiological conditions, including synaptic pruning (Schafer et al, 2012; Um, 2017), but are important during neurodegenerative diseases (Hirsch and Hunot, 2009; Rayaprolu et al, 2013; Nalls et al, 2014; Ulrich et al, 2014; Sims et al, 2017; Kang et al, 2018b). The process for expansion and transformation of iPS cells into microglia cells is a laborious and complicated procedure, with several different protocols to follow in the literature (Muffat et al, 2016; Brownjohn et al, 2018)

Methods

Results

Conclusion