Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches
Summary
This manuscript demonstrates the depletion of gene expression in the midgut of the German cockroach through oral ingestion of double-stranded RNA encapsulated in liposomes.
Abstract
RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool operating by disruption of essential gene expression. Although different methods, such as injection, feeding, and soaking, have been reported for successful delivery of double-stranded RNA (dsRNA), the efficiency of RNAi through oral delivery of dsRNA is highly variable among different insect groups. The German cockroach, Blattella germanica, is highly sensitive to the injection of dsRNA, as shown by many studies published previously. The present study describes a method to demonstrate that the dsRNA encapsulated with liposome carriers is sufficient to retard the degradation of dsRNA by midgut juice. Notably, the continuous feeding of dsRNA encapsulated by liposomes significantly reduces the tubulin expression in the midgut, and led to the death of cockroaches. In conclusion, the formulation and utilization of dsRNA lipoplexes, which protect dsRNA against nucleases, could be a practical use of RNAi for insect pest control in the future.
Introduction
RNAi has been demonstrated as an effective method to knockdown gene expression through a mechanism of a post-transcriptional silencing pathway triggered by dsRNA molecules in many eukaryotes1. Over the past decade of study, RNAi has become a useful tool to study the functions of genes from development to behavior by depleting the expression of specific genes via injection and/or feeding of dsRNA in various taxa of insects2,3. Due to the specificity and robustness of the depleting effect, the application of RNAi is currently being considered as a potential strategy for pest control management4,5. However, the efficiency of RNAi varies widely between insect species, depending on the different genes being targeted and the delivery methods. A growing body of evidence suggests that the instability of dsRNA, which is degraded by ribonucleases, is a critical factor in the limited efficacy of RNAi5,6. For instance, the low RNAi sensitivity in Manduca sexta has been explained by the fact that the dsRNA mixed with hemolymph was quickly degraded within 1 hour7. Similarly, the presence of alkaline nucleases in the midgut, which efficiently degrade ingested dsRNA, is strongly correlated with low RNAi efficiency in different insect orders8,9,10.
The oral delivery of dsRNA is particularly interesting for the application of RNAi in a pest control strategy, but a method to retard the degradation of dsRNA by the nucleases in the midgut has not yet been developed, which would have the potential to ensure effective RNAi through feeding. However, the unresponsiveness of RNAi to oral delivery of dsRNA has been reported by feeding large amount of dsRNA, e.g. 50 µg in Bombyx mori, or continuously feeding for 8 days (8 µg dsRNA in total) in the locust species. The German cockroach, Blattella germinica, is highly sensitive to RNAi by the injection of dsRNA11,12,13,14, but is not responsive to dsRNA through feeding. Recently, Lin et al. (2017) have demonstrated that the dsRNA encapsulated with liposome carriers results in successful RNAi to knockdown the α-tubulin gene expression in the midgut and trigger significant mortality of the German cockroach15. As the degradation of dsRNA in the midgut is the limiting factor for oral RNAi, the liposome carriers serve as a vehicle to protect dsRNA from degradation, which is readily applicable in other insects with strong nuclease activities in the gut. Of note, the reason for choosing the particular transfection reagent (see Table of Materials) we used as liposome carrier in the current protocol is that it has been tested for insect cell line transfection with less toxicity, according to the manufacturer's instructions. According to the comparison of different liposome transfection systems in Gharavi et al. (2013)16, the efficiency of transfecting small interfering RNA (siRNA) is approximately the same between this and other commercially available systems that have been used for dsRNA delivery systems in other insects17,18.Furthermore, our feeding method is careful enough to ensure the proper amount of dsRNA is ingested by each cockroach, and that the results are robust and confirmed. In summary, the present protocol and results demonstrate that using dsRNA lipoplexes improves dsRNA stability and opens the door to the design of the strategy oral delivery of RNAi, which is a promising approach for pest control in the future.
Protocol
Representative Results
Discussion
This protocol presents a method for effective RNAi through oral delivery of dsRNA lipoplexes, involving protection against ribonuclease digestion in the midgut juice of the German cockroach. As shown in other studies in various insect species, the poor RNAi effect through oral delivery of dsRNA is mostly accounted for by the degradation of dsRNA8,9,10. This protocol produces liposomes that serve as protective vehicles in oral de…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This study was supported by grants from Taiwan (Ministry of Science and Technology, MOST 100-2923-B-002-002-MY3 and 106-2313-B-002-011-MY3 to H.J.L.), the Czech Republic (Grant agency of South Bohemia University, GAJU grant 065/2017/P to Y.H.L), and Spain (Spanish Ministry of Economy and Competitiveness, grants CGL2012-36251 and CGL2015-64727-P to X.B., and the Catalan Government, grant 2014 SGR 619 to X.B.); it also received financial support from the European Fund for Economic and Regional Development (FEDER funds to X.B.).
Materials
| GenJe Plus DNA in vitro Transfection reagent | SignaGen | SL100499 | for lipoplexes preparation |
| Blend Taq plus | TOYOBO | BTQ-201 | for PCR |
| Fast SYBR Green Master Mix | ABI | 4385612 | for qPCR |
| FirstChoice RLM-RACE Kit | Invitrogen | AM1700 | for 3' UTR identification |
| MEGAscript T7 Transcription Kit | Invitrogen | AMB13345 | for dsRNA synthesis |
| TURBO DNase | Invitrogen | AM2239 | remove DNA template from dsRNA |
| TRIzol | Invitrogen | 15596018 | for dsRNA or total RNA extraction |
| RQ1 RNase-Free Dnase | Promega | M6101 | remove DNA template from total RNA |
| chloroform | Sigma-Aldrich | C2432 | for dsRNA or total RNA extraction |
| 2-Propanol | Sigma-Aldrich | I9516 | for dsRNA or total RNA extraction |
| ethanol | Sigma-Aldrich | 24102 | for dsRNA or total RNA extraction |
| Diethyl pyrocarbonate, DEPC | Sigma-Aldrich | D5758 | for RNase free water preparation |
| glucose solution | Sigma-Aldrich | G3285 | for lipoplexes preparation |
| Sodium chloride, NaCl | Sigma-Aldrich | S7653 | insect saline buffer formula |
| Potassium chloride, KCl | Sigma-Aldrich | P9333 | insect saline buffer formula |
| Calcium chloride, CaCl2 | Sigma-Aldrich | C1016 | insect saline buffer formula |
| Magnesium chloride hexahydrate, MgCl2.6H2O | Sigma-Aldrich | M2670 | insect saline buffer formula |
| EGTA | Sigma-Aldrich | E3889 | enzyme inhibitor |
| dissecting scissor | F.S.T. | cockroach dissection | |
| fine tweezers | F.S.T. | cockroach dissection | |
| flexible tweezer | F.S.T. | cockroach holding | |
| pipetman | RAININ | P10 | sample preparation |
| microcentrifuge tube | Axygen | MCT175C, PCR02C | sample preparation |
| pipette tip | Axygen | sample preparation | |
| vortexter | Digisystem | vm1000 | sample preparation |
| Minispin centrifuge | The Gruffin Group | GMC 206 | for liquid spin down |
| Centrifuge | ALC | PK121R | sample preparation |
| pH meter | JENCO | 6071 | for pH adjust |
| micro-volume spectrophotometer | Quawell | Q3000 | nucleic acid quantitative |
| PCR Thermal cycler | ABI | 2720 | for template PCR or dsRNA synthesis incubation |
| quantitative real-time PCR | ABI | StepOne plus | gene expression quantitative |
| Centrifugal Vacuum Concentrators | eppendorf | 5301 | for dsRNA or total RNA extraction |
| Multipette | eppendorf | xstream | for real-time PCR sample loading |
| Agarose I | amresco | 0710 | for nucleic acid electrophoresis |
| tub gene specfifc forward preimer | tri-I biotech | GGG ACA AGC CGG AGT GCA GA | |
| tub gene specfifc reverse preimer | tri-I biotech | TCC TGC TCC TGT CTC GCT GA | |
| dsTub template forward primer | tri-I biotech | TAA TAC GAC TCA CTA TAG GGA CAA GCC GGA GTG CAG | |
| dsTub template reverse primer | tri-I biotech | TAA TAC GAC TCA CTA TAG GGT CCT GCT CCT GTC TCG CTG | |
| dsEGFP template forward preimer | tri-I biotech | TAA TAC GAC TCA CTA TAG GGT ATG GTG AGC AAG GGC GAG GAG | |
| dsEGFP template reverse preimer | tri-I biotech | TAA TAC GAC TCA CTA TAG GGT GGC GGA TCT TGA AGT TCA CC | |
| tub qPCR forward primer | tri-I biotech | GGA CCG CAT CAG GAA ACT GGC | |
| tub qPCR reverse preimer | tri-I biotech | CCA CAG ACA GCC TCT CCA TGA GC | |
| ef1 qPCR forward primer | tri-I biotech | CGC TTG AGG AAA TCA AGA AGG A | |
| ef1 qPCRreverse preimer | tri-I biotech | CCT GCA GAG GAA GAC GAA G |
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