<meta charset="utf8"></head><body>Nanopartikel-vermittelte dsRNA-Fütterung für Gen-Silencing in Seidenraupenlarven

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rna-based+antiviral+immunity.">Rna-based antiviral immunity.</a> Nat Rev Immunol. 10 (9), 632-644 (2010).</li><li>Yu, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+dsRNA+for+RNAi+in+insects:+An+overview+and+future+directions.">Delivery of dsRNA for RNAi in insects: An overview and future directions.</a> Insect Sci. 20 (1), 4-14 (2013).</li><li>Liang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+of+the+immune+gene+bmpgrp-l4+in+the+midgut+affects+the+growth+of+silkworm+(bombyx+mori)+larvae.">Silencing of the immune gene bmpgrp-l4 in the midgut affects the growth of silkworm (bombyx mori) larvae.</a> Insect Mol Biol. 32 (4), 340-351 (2023).</li><li>Tian, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+control+of+a+lepidopteran+pest+Spodoptera+exigua+by+ingestion+of+bacteria+expressing+dsrna+of+a+non-midgut+gene.">Developmental control of a lepidopteran pest Spodoptera exigua by ingestion of bacteria expressing dsrna of a non-midgut gene.</a> PLoS One. 4 (7), e6225 (2009).</li><li>Kamath, R. 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R., Choong, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+chitosan+formulations+in+cancer+therapy.">The use of chitosan formulations in cancer therapy.</a> J Microencapsul. 25 (4), 275-279 (2008).</li><li>Zhang, X., Zhang, J., Zhu, K. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitosan/double-stranded+RNA+nanoparticle-mediated+RNA+interference+to+silence+chitin+synthase+genes+through+larval+feeding+in+the+African+malaria+mosquito+(Anopheles+gambiae).">Chitosan/double-stranded RNA nanoparticle-mediated RNA interference to silence chitin synthase genes through larval feeding in the African malaria mosquito (Anopheles gambiae).</a> Insect Mol Biol. 19 (5), 683-693 (2010).</li><li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitosan/interfering+rna+nanoparticle+mediated+gene+silencing+in+disease+vector+mosquito+larvae.">Chitosan/interfering rna nanoparticle mediated gene silencing in disease vector mosquito larvae.</a> J Vis Exp. (97), e52523 (2015).</li><li>Sandal, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle-shielded+dsrna+delivery+for+enhancing+rnai+efficiency+in+cotton+spotted+bollworm+Earias+vittella+(lepidoptera:+Nolidae).">Nanoparticle-shielded dsrna delivery for enhancing rnai efficiency in cotton spotted bollworm Earias vittella (lepidoptera: Nolidae).</a> Int J Mol Sci. 24 (11), 9161 (2023).</li><li>Zhou, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitosan/dsrna+polyplex+nanoparticles+advance+environmental+rna+interference+efficiency+through+activating+clathrin-dependent+endocytosis.">Chitosan/dsrna polyplex nanoparticles advance environmental rna interference efficiency through activating clathrin-dependent endocytosis.</a> Int J Biol Macromol. 253 (Pt 4), 127021 (2023).</li><li>Liu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+function+of+bombyx+toll9-2+in+the+silkworm+(Bombyx+mori)+larval+midgut:+Activation+by+escherichia+coli/lipopolysaccharide+and+regulation+of+growth.">Immunological function of bombyx toll9-2 in the silkworm (Bombyx mori) larval midgut: Activation by escherichia coli/lipopolysaccharide and regulation of growth.</a> Arch Insect Biochem Physiol. 116 (4), e22130 (2024).</li><li>Cheng, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+silkworm+larvae+growth+and+properties+of+silk+fibres+after+direct+feeding+of+copper+or+silver+nanoparticles.">Characterization of silkworm larvae growth and properties of silk fibres after direct feeding of copper or silver nanoparticles.</a> Mater Design. 129, 125-134 (2017).</li><li>Wang, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+efficacy+of+rnai+mediated+by+various+nanoparticles+in+the+rice+striped+stem+borer+(Chilo+suppressalis).">Comparison of efficacy of rnai mediated by various nanoparticles in the rice striped stem borer (Chilo suppressalis).</a> Pestic Biochem Physiol. 165, 104467 (2020).</li><li>Gurusamy, D., Mogilicherla, K., Palli, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitosan+nanoparticles+help+double-stranded+rna+escape+from+endosomes+and+improve+rna+interference+in+the+fall+armyworm,+Spodoptera+frugiperda.">Chitosan nanoparticles help double-stranded rna escape from endosomes and improve rna interference in the fall armyworm, Spodoptera frugiperda.</a> Arch Insect Biochem Physiol. 104 (4), e21677 (2020).</li><li>Qi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+design+of+ros+scavenging+and+fluorescent+gold+nanoparticles+to+deliver+sirna+to+improve+plant+resistance+to+Pseudomonas+syringae.">Rational design of ros scavenging and fluorescent gold nanoparticles to deliver sirna to improve plant resistance to Pseudomonas syringae.</a> J Nanobiotechnol. 22 (1), 446 (2024).</li><li>Laisney, J., Loczenski Rose, V., Watters, K., Donohue, K. 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<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL centrifuge tube</td>
			<td>Sangon</td>
			<td>F601620</td>
			<td>for dsRNA or nanoparticles reaction</td>
		</tr>
		<tr>
			<td>10 &#956;l pipette</td>
			<td>Eppendorf</td>
			<td>P13473G</td>
			<td>to aspirate or resuspend liquid</td>
		</tr>
		<tr>
			<td>100 &#956;l pipette</td>
			<td>Eppendorf</td>
			<td>Q12115G</td>
			<td>to aspirate or resuspend liquid</td>
		</tr>
		<tr>
			<td>2.5 &#956;l pipette</td>
			<td>Eppendorf</td>
			<td>P20777G</td>
			<td>to aspirate or resuspend liquid</td>
		</tr>
		<tr>
			<td>20 &#956;l pipette</td>
			<td>Eppendorf</td>
			<td>H19229E</td>
			<td>to aspirate or resuspend liquid</td>
		</tr>
		<tr>
			<td>200 &#956;l pipette</td>
			<td>Eppendorf</td>
			<td>H20588E</td>
			<td>to aspirate or resuspend liquid</td>
		</tr>
		<tr>
			<td>6-well Clear TC-treated Multiple Well Plates</td>
			<td>Costar</td>
			<td>3516</td>
			<td>for silkworm rearing individually</td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Aladdin</td>
			<td>A116165</td>
			<td>to make TAE</td>
		</tr>
		<tr>
			<td>Agarose M</td>
			<td>BBI Life Sciences</td>
			<td>A610013</td>
			<td>for agarose gel electrophosis</td>
		</tr>
		<tr>
			<td>Analytical balance</td>
			<td>Sartorius</td>
			<td>BSA224S</td>
			<td>to weight ingredients</td>
		</tr>
		<tr>
			<td>Centrifuge&#160;</td>
			<td>Sartorius</td>
			<td>Centrisart A-14C</td>
			<td>to centrifuge to form dsRNA or nanoparticles</td>
		</tr>
		<tr>
			<td>Chitosan</td>
			<td>Sigma-Aldrich</td>
			<td>C3646</td>
			<td>to combine with dsRNA for preparation of nanoparticles</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sangon</td>
			<td>A500895</td>
			<td>to make TAE</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Aladdin</td>
			<td>E130059</td>
			<td>to make TAE, or for dsRNA precipitation</td>
		</tr>
		<tr>
			<td>Freezer</td>
			<td>Siemens</td>
			<td>iQ300</td>
			<td>to store dsRNA or nanoparticles</td>
		</tr>
		<tr>
			<td>GoTaq Green Master Mix</td>
			<td>Promega</td>
			<td>M712</td>
			<td>for PCR reaction</td>
		</tr>
		<tr>
			<td>GoTaq qPCR Master Mix</td>
			<td>Promega</td>
			<td>A6002</td>
			<td>for qRT-PCR reaction</td>
		</tr>
		<tr>
			<td>Isopropanol&#160;</td>
			<td>Aladdin</td>
			<td>I112011</td>
			<td>for dsRNA precipitation</td>
		</tr>
		<tr>
			<td>NanoDrop Microvolume UV-Vis Spectrophotometer</td>
			<td>ThermoFisher</td>
			<td>One</td>
			<td>to determine the concentration of dsRNA</td>
		</tr>
		<tr>
			<td>ph meter</td>
			<td>Sartorius</td>
			<td>&#160;PB-10</td>
			<td>to prepare buffers</td>
		</tr>
		<tr>
			<td>SanPrep Column PCR Product Purification Kit</td>
			<td>Sangon</td>
			<td>B518141</td>
			<td>for PCR product purification</td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Sigma-Aldrich</td>
			<td>S2889</td>
			<td>to make 100 mM sodium acetate buffer</td>
		</tr>
		<tr>
			<td>Sodium sulfate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>239313</td>
			<td>to make 100 mM sodium sulfate buffer</td>
		</tr>
		<tr>
			<td>T7 RiboMAX Express RNAi System</td>
			<td>Promega</td>
			<td>P1700</td>
			<td>for dsRNA synthesis</td>
		</tr>
		<tr>
			<td>ThermoMixer</td>
			<td>Eppendorf</td>
			<td>C</td>
			<td>for dsRNA generation or nanoparticles heating</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Sangon</td>
			<td>A501492</td>
			<td>to make TAE</td>
		</tr>
		<tr>
			<td>Vortex&#160;</td>
			<td>IKA</td>
			<td>Vortex 3</td>
			<td>to prepare chitosan/dsRNA nanoparticles</td>
		</tr>
	</tbody>
</table>

larval rna interference in silkworm bombyx mori through chitosan/dsrna nanoparticle delivery

The silkworm, Bombyx mori, is an insect domesticated more than 5000 years ago in China. Due to its ability to produce silk, the silkworm is an important economic insect in Chinese agriculture and sericulture. The silkworm is second only to the fruit fly as the model insect. As a model insect in Lepidoptera, the silkworm is easy to rear, with a large body size and plenty of mutants. Meanwhile, the silkworm is the first Lepidopteran insect with its complete genome sequenced1. A lot of databases providing information for genome2, transcriptome3, expressed sequence tag (EST)4, non-coding RNA5, and microsatellite6 are also available to the public. The above facts make the silkworm a perfect model for genetic research.
RNA interference (RNAi) is a cellular process in which double-stranded RNA (dsRNA) molecules bind and slice the complementary messenger RNA (mRNA), thereby achieving the silencing effect of the target gene. This mechanism is naturally present in bacteria to defend against the invasion of viruses7. Later, it was found that RNAi is conserved in animals, plants, and microbes. Due to its powerful sequence-specific silencing effect, RNAi is used in fundamental research to manipulate gene expression and study gene function. RNAi is achieved through the delivery of dsRNA into cells.
In insects, there are three common ways to deliver dsRNA, which are microinjection, feeding, and soaking8. At the moment, successful RNAi reports in the silkworms through naked dsRNA delivery are conducted by dsRNA injection9. The advantages of microinjection are the immediate delivery of dsRNA into the hemolymph and precise dsRNA amount control. However, certain disadvantages of microinjection also exist. For example, it is time-consuming, and it requires delicate devices. It is also important to optimize the injection needles, injection volume, and dsRNA amount. Therefore, an alternative way to deliver dsRNA to silkworms becomes necessary. Because an insect&#39;s exoskeleton is a water-tight barrier that is made of chitin, soaking insect larvae to achieve RNAi is rarely reported, which is not a good option for RNAi in insects. Feeding of dsRNA is labor-saving, cost-effective, and easy to perform10. This method is also applicable for high-throughput gene screening11. However, it is found that a DNA/RNA non-specific nuclease, namely BmdsRNase, is present in the midgut and midgut juice of the silkworm larvae12. This nuclease is shown to digest dsRNA, preferably13. Therefore, feeding naked dsRNA to the silkworm to silence the gene expression seems to be difficult.
Recently, nanoparticle-shielded dsRNA is proved to be a good alternative to increase the RNAi efficiency by feeding14. Chitosan is an inexpensive, nontoxic, and biodegradable polymer, which can be prepared by deacetylation of chitin, a naturally occurring and the second most abundant biopolymer after cellulose15. Because the amino group in the chitosan is positively charged and the phosphate group on the backbone of the dsRNA is negatively charged, the chitosan/dsRNA nanoparticles could be formed by self-assembly of polycations16. Chitosan/dsRNA nanoparticles are effective in achieving RNAi through larval feeding in mosquitos Aedes aegypti and Anopheles gambiae17, cotton spotted bollworm Earias vittella18 and carmine spider mite Tetranychus cinnabarinus19.
In order to develop a methodology for dsRNA delivery by feeding in silkworms to gain successful RNAi efficiency, this report focuses on describing step-by-step procedures on how to prepare the chitosan/dsRNA nanoparticles and feed the nanoparticles to the silkworm larvae. This methodology is relatively inexpensive, labor-saving and easy to follow, which can be adapted for gene silencing studies in other insects. We aim to provide an easier protocol for the Lepidopteran dsRNA delivery method with higher RNAi efficiency.

<meta charset="utf8"></head><body>Autor im Rampenlicht: Förderung der Gen-Silencing-Forschung bei Seidenraupen mit dsRNA-Verabreichung durch Fütterung von Chitosan-Nanopartikeln

Larvale RNA-Interferenz in der Seidenraupe Bombyx mori durch Verabreichung von Chitosan/dsRNA-Nanopartikeln

The silkworm, Bombyx mori, is an important economic insect with thousands of years of history in China. Meanwhile, the silkworm is the model insect of ...

larval-rna-interference-silkworm-bombyx-mori-through-chitosandsrna

Research

JoVE Journal

Biology

Larval RNA Interference in Silkworm Bombyx mori through Chitosan/dsRNA Nanoparticle Delivery

1.1K Aufrufe.  Guangzhou University. Ziel dieser Forschung ist es, eine Methode zu entwickeln, um die Larven der Seidenraupe Bombyx mori mit dsRNA zu füttern, was zu einem Gen-Silencing-Effekt führt. Dies wird bei Studien zur Genregulation und beim Hochdurchsatz-Genscreening hilfreich sein. Chitosan ist ein preiswertes, ungiftiges und biologisch abbaubares Polymer.Es wurde festgestellt, dass Chitosan/dsRNA-Nanopartikel den RNAi-Effekt wirksam verursachen, indem sie die Mücken füttern....

Serie: Autor im Rampenlicht: Förderung der Gen-Silencing-Forschung bei Seidenraupen mit dsRNA-Verabreichung durch Fütterung von Chitosan-Nanopartikeln

The silkworm, Bombyx mori, is an important economic insect with thousands of years of history in China. Meanwhile, the silkworm is the model insect of Lepidoptera with a good accumulation of basic research. It is also the first insect in Lepidoptera with its complete genome sequenced and assembled, which provides a solid foundation for gene functional study. Although RNA interference (RNAi) is widely used in reverse gene functional study, it is refractory in silkworms and other Lepidopteran species. Previous successful RNAi-related research to deliver double-stranded RNA (dsRNA) was performed through injection only. Delivery of dsRNA through feeding is never reported. In this article, we describe step-by-step procedures to prepare the chitosan/dsRNA nanoparticles, which are fed to the silkworm larvae by ingestion. The protocol includes (i) selection of the proper stage of silkworm larvae, (ii) synthesis of dsRNA, (iii) preparation of the chitosan/dsRNA nanoparticles, and (iv) feeding the silkworm larvae with chitosan/dsRNA nanoparticles. Representative results, including gene transcript confirmation and phenotype observation, are presented. dsRNA feeding is a simple technique for RNAi in silkworm larvae. Since silkworm larvae are easy to rear and large enough to operate, it provides a good model to demonstrate larval RNAi in insects. In addition, the simplicity of this technique stimulates more student involvement in research, making silkworm larvae an ideal genetic system for use in a classroom setting.

Presented here is a protocol for chitosan/dsRNA nanoparticle delivery in silkworm Bombyx mori larvae to induce gene silencing through ingestion.