An efficient method for gene knock‐down by RNA interference in human skin mast cells

Abstract

Mast cells (MCs) from human skin have been notoriously resistant to gene manipulation, and a method to knock‐down gene expression in in situ differentiated MCs is highly desired. The Dharmacon Accell® transfection system proved successful on several “difficult‐to‐transfect” cells. In the present work, we therefore tested this method on skin‐derived MCs using different siRNA entities. The siRNA was readily taken up, followed by pronounced, specific reduction of gene and protein expression. Hence, we present the first efficient technique for the manipulation of gene expression in primary skin MCs ex vivo, which combines high transfection rates with retained cell viability.

Yin-Yang of IL-33 in Human Skin Mast Cells: Reduced Degranulation, but Augmented Histamine Synthesis through p38 Activation

“…Specific inhibitors of both kinases revealed that p38, but not JNK, was implicated in the regulation of HDC and histamine by IL-33. We recently established the method of Accell-assisted RNA interference as a knock-down method in hard-to-transfect tissue-derived MCs (Hazzan et al, 2017b). By eliminating JNK and p38 expression, we confirmed that p38, but not JNK was implicated in HDC and histamine regulation.…”

“…Accell-mediated small interfering RNA (siRNA) selfdelivery has recently been introduced as a method to perturb endogenous levels of proteins in tissue MCs (Hazzan et al, 2017a(Hazzan et al, , 2017b. Exploiting this advancement, we employed JNK1-specific and p38a-specific RNA interference to eliminate their expression after verification that these were the major isoforms expressed in skin MCs (Motakis et al, 2014).…”

“…Accell-mediated RNA interference RNA interference in MCs was performed according to a recently established protocol (Hazzan et al, 2017a(Hazzan et al, , 2017b using the Accell siRNA technology (Dharmacon, Lafayette, CO). MCs were transfected by gene-targeting siRNA or non-targeting siRNA (each at 1 mM for 48 hours) in Accell medium (supplemented with nonessential amino acids and L-glutamine).…”

IL-33 and MRGPRX2-Triggered Activation of Human Skin Mast Cells—Elimination of Receptor Expression on Chronic Exposure, but Reinforced Degranulation on Acute Priming

“…RNA interference in MCs was performed according to an established protocol [24,35,36] using the Accell ® siRNA technology (Dharmacon, Lafayette, CO, USA). Briefly, MCs were washed with 1 × Accell siRNA medium (supplemented with Non-Essential Amino Acids and L-Glutamine), plated at 1 × 10 6 /mL in Accell siRNA medium and treated with 1 µM JNK-targeting siRNA (E-003514-00-0010) or non-targeting siRNA (D-001910-10-50, serving as control) for 48 h. After incubation, cells were treated with or without IL-33 for the times indicated.…”

Transduction and transfection of difficult‐to‐transfect cells: Systematic attempts for the transfection of protozoa Leishmania

“…endothelial cells, hematopoietic cells, and hippocampal and cortical neurons), [3][4][5][6][7] mast cells, 8 and natural killer cells. 1,9 Ongoing experiments with siRNA indicate that macrophages also belong to these cell types (unpublished result).…”