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Comparative Study
, 158 (3-4), 224-32

Comparison of Small Interfering RNA (siRNA) Delivery Into Bovine Monocyte-Derived Macrophages by Transfection and Electroporation

Affiliations
Comparative Study

Comparison of Small Interfering RNA (siRNA) Delivery Into Bovine Monocyte-Derived Macrophages by Transfection and Electroporation

Kirsty Jensen et al. Vet Immunol Immunopathol.

Abstract

The manipulation of the RNA interference pathway using small interfering RNA (siRNA) has become the most frequently used gene silencing method. However, siRNA delivery into primary cells, especially primary macrophages, is often considered challenging. Here we report the investigation of the suitability of two methodologies: transient transfection and electroporation, to deliver siRNA targeted against the putative immunomodulatory gene Mediterranean fever (MEFV) into primary bovine monocyte-derived macrophages (bMDM). Eleven commercial transfection reagents were investigated with variable results with respect to siRNA uptake, target gene knock-down, cell toxicity and type I interferon (IFN) response induction. Three transfection reagents: Lipofectamine 2000, Lipofectamine RNAiMAX and DharmaFECT 3, were found to consistently give the best results. However, all the transfection reagents tested induced an IFN response in the absence of siRNA, which could be minimized by reducing the transfection reagent incubation period. In addition, optimized siRNA delivery into bMDM by electroporation achieved comparable levels of target gene knock-down as transient transfection, without a detectable IFN response, but with higher levels of cell toxicity. The optimized transient transfection and electroporation methodologies may provide a starting point for optimizing siRNA delivery into macrophages derived from other species or other cells considered difficult to investigate with siRNA.

Keywords: Bovine; Electroporation; Macrophage; Transfection; siRNA.

Figures

Fig. 1
Fig. 1
Comparison of the efficacy of eleven commercially available reagents to transfect siRNA into bovine monocyte-derived macrophages (bMDM). bMDM were transfected with 50 nM FITC-labelled siRNA using three different concentrations of the transfection reagents HiPerFect (HPT), INTERFERin (INT), Lipofectamine RNAiMAX (RNA), Lipofectamine 2000 (L2K), siPORT Amine (SPA), X-tremeGENE (XTG), N-TER (NTR) and DharmaFECT 1 (DF1), DharmaFECT 2 (DF2), DharmaFECT 3 (DF3) and DharmaFECT 4 (DF4) following the manufacturer's instructions. In addition bMDM were cultured with 50 nM FITC-labelled siRNA in the absence of transfection reagents as a control (C). After 16 h the percentage of bMDM transfected with FITC-siRNA (grey bars) and the percentage of viable bMDM (black diamonds) were measured by flow cytometry. The result is representative of three repeat experiments using bMDM from different animals.
Fig. 2
Fig. 2
Transient transfection reagents induce a type I interferon (IFN)-response in bovine monocyte-derived macrophages (bMDM) in the absence of siRNA. The siRNA uptake efficacy and off-target effects of five transfection reagents, INTERFERin (INT), Lipofectamine RNAiMAX (RNA), Lipofectamine 2000 (L2K), X-tremeGENE (XTG) and DharmaFECT 3 (DF3) were investigated. (A) bMDM were cultured with pre-determined concentrations of the transfection reagents in the absence of siRNA for 48 h (48 h incubation) or the medium was changed 24 h post transfection reagent treatment and cultured for a further 24 h (24 h incubation). The mRNA levels of the type I IFN-response gene interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) were measured by qRT-PCR. The results are expressed as the IFIT1 mRNA fold difference compared to that measured in untreated cells (NC). Error bars denote the variation between three technical replicates. (B) bMDM were transfected with MEFV siRNA using the five transfection reagents for 24 h before the medium was replaced. MEFV mRNA levels in bMDM transfected with (NTC) and without (TC) non-target control siRNA using Lipofectamine RNAiMAX were also investigated. After 48 h the bMDM were activated with 100 ng/ml LPS for 2 h. The results are expressed as the percentage MEFV mRNA detected compared to that measured in NC. Error bars denote the variation between three technical replicates. The results are representative of three repeat experiments using bMDM from different animals.
Fig. 3
Fig. 3
Optimization of siRNA uptake by electroporation. The ability of bovine monocyte-derived macrophages (bMDM) in different buffers, Opti-MEM and siPORT siRNA electroporation buffer, to take up 1 μM FITC-labelled siRNA after electroporation at various voltage and capacitance settings was compared. After 16 h the percentage of bMDM transfected with FITC-siRNA (grey bars) and the percentage of viable bMDM (black diamonds) were measured by flow cytometry. The result is representative of three repeat experiments using bMDM from different animals.
Fig. 4
Fig. 4
Electroporation is an effective method for siRNA uptake in bovine monocyte-derived macrophages (bMDM) without inducing a type I interferon (IFN) response. bMDM were electroporated without siRNA (EC), with MEFV siRNA or non-target control siRNA (NTC siRNA). (A) After 48 h the electroporated bMDM were activated with 100 ng/ml LPS for 2 h. The results are expressed as the percentage MEFV mRNA detected compared to that measured in untreated cells (NC). Error bars denote the variation between three technical replicates. (B) After 48 h cells were harvested and mRNA levels of the IFN-response gene interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) were measured by qRT-PCR. The results are expressed as the IFIT1 mRNA fold difference compared to that measured in NC. Error bars denote the variation between three technical replicates. The results are representative of three repeat experiments using bMDM from different animals.

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