Gene silencing in the aedine cell lines C6/36 and U4.4 using long double-stranded RNA

Abstract

Background: RNA interference (RNAi) is a target-specific gene silencing method that can be used to determine gene functions and investigate host-pathogen interactions, as well as facilitating the development of ecofriendly pesticides. Commercially available transfection reagents (TRs) can improve the efficacy of RNAi. However, we currently lack a product and protocol for the transfection of insect cell lines with long double-stranded RNA (dsRNA).

Methods: We used agarose gel electrophoresis to determine the capacity of eight TRs to form complexes with long dsRNA. A CellTiter-Glo assay was then used to assess the cytotoxicity of the resulting lipoplexes. We also measured the cellular uptake of dsRNA by fluorescence microscopy using the fluorophore Cy3 as a label. Finally, we analyzed the TRs based on their transfection efficacy and compared the RNAi responses of Aedes albopictus C6/36 and U4.4 cells by knocking down an mCherry reporter Semliki Forest virus in both cell lines.

Results: The TRs from Biontex (K4, Metafectene Pro, and Metafectene SI+) showed the best complexing capacity and the lowest dsRNA:TR ratio needed for complete complex formation. Only HiPerFect was unable to complex the dsRNA completely, even at a ratio of 1:9. Most of the complexes containing mCherry-dsRNA were nontoxic at 2 ng/µL, but Lipofectamine 2000 was toxic at 1 ng/µL in U4.4 cells and at 2 ng/µL in C6/36 cells. The transfection of U4.4 cells with mCherry-dsRNA/TR complexes achieved significant knockdown of the virus reporter. Comparison of the RNAi response in C6/36 and U4.4 cells suggested that C6/36 cells lack the antiviral RNAi response because there was no significant knockdown of the virus reporter in any of the treatments.

Conclusions: C6/36 cells have an impaired RNAi response as previously reported. This investigation provides valuable information for future RNAi experiments by showing how to mitigate the adverse effects attributed to TRs. This will facilitate the judicious selection of TRs and transfection conditions conducive to RNAi research in mosquitoes.

Keywords: Aedes albopictus; Arbovirus; Cell culture; RNAi; Semliki Forest virus; Transfection reagents; Vector; dsRNA; mCherry.

Fig. 1

Cytotoxicity of dsRNA complexed with five transfection reagents in Ae. albopictus (a) C6/36 and (b) U4.4 cells. The cells were treated at ~50% confluency with only the TR or with (1) 50, (2) 100, and (3) 200 ng of the complexes of TRs and mCherry-dsRNA per well (n = 6). Cell viability was determined using a CellTiter-Glo assay. The data were normalized to the untreated control (treatment/control × 100) and the mean cell viability is displayed, with error bars representing coefficient of variation (both in %). The untreated control is represented as 0. The dotted line represents the toxicity cutoff set at 80%. Abbreviations: K4 K4 transfection system, MPro Metafectene Pro, MSI+ Metafectene SI+, Lipo Lipofectamine 2000, CellFect CellFectin II

Fig. 2

Uptake of Cy3-labeled dsRNA into C6/36 cells 24 h post-transfection. a Mean fluorescence of Cy3 signal from the labeled dsRNA and (b) mean fluorescence of Hoechst 33342 (both n = 3, with error bars indicating standard deviations). c–h Fluorescence images taken at 20× magnification. c Labeled dsRNA was applied without transfection reagent (w/o). d–h Cells were transfected with the labeled dsRNA using (d) K4, (e) Metafectene Pro, (f) Metafectene SI+, (g) Lipofectamine 2000, and (h) CellFectin II. The TRs were used to transfect cells with 200 ng labeled dsRNA, except Lipofectamine 2000 with only 50 ng. The cell nuclei are stained in blue and the labeled dsRNA in red. The percentage of transfected cells per image for all conditions can be found in Table S2

Fig. 3

Uptake of Cy3-labeled dsRNA into U4.4 cells 24 h post-transfection. a Mean fluorescence of Cy3 signal from the labeled dsRNA and (b) mean fluorescence of Hoechst 33342 (both n = 3, with error bars indicating standard deviations). c–h Fluorescence images taken at 20× magnification. c Labeled dsRNA was applied without transfection reagent (w/o). d–h Cells were transfected with the labeled dsRNA using (d) K4, (e) Metafectene Pro, (f) Metafectene SI+, (g) Lipofectamine 2000, and (h) CellFectin II. The TRs were used to transfect cells with 200 ng labeled dsRNA, except Lipofectamine 2000 with only 50 ng. The cell nuclei are stained in blue and the labeled dsRNA in red. The percentage of transfected cells per image for all conditions can be found in Table S2

Fig. 4

Knockdown of virus reporter mCherry-SFV in (a) C6/36 and (b) U4.4 cells. Both cell lines were treated with 0.5 ng/µL dsRNA targeting GFP or mCherry (mCh). The dsRNAs were introduced into the cells using K4, Metafectene Pro (MPro), Metafectene SI+ (MSI), Lipofectamine 2000 (Lipo), or CellFectin II (CellFect). At 24 hpt, cells were infected with mCherry-SFV. The total intensity and cell count were analyzed at 30 (for C6/36) and 56 (for U4.4) hpi. The data are mean values (n = 3) of the total red intensity per cell (total red intensity/total cell count) and the error bars represent standard deviations. Statistical significance was determined by ANOVA and Šidák’s multiple comparison test (****P < 0.0001, ns = P > 0.05)