Name: practical use of rna interference: oral delivery of double-stranded rna in liposome carriers for cockroaches
Uploaded: 2018-05-01T14:45:00
Description: Watch this Scientific Journal Video about Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches at JoVE.com

Denne undersøgelse blev støttet af tilskud fra Taiwan (Ministeriet for videnskab og teknologi, mest 100-2923-B-002-002-MY3 og 106-2313-B-002-011-MY3 til H.J.L.), Tjekkiet (giver agenturet i Sydböhmen Universitet, Andreas give Y.H.L 2017-065-P), og Spanien ( Spanske Ministeriet for økonomi og konkurrenceevne, giver CGL2012-36251 og CGL2015-64727-P til X.B. og den catalanske regering, give 2014 SGR 619 til X.B.); Det har også modtaget økonomisk støtte fra den europæiske fond for økonomisk og Regionaludvikling (EFRU-midler til X.B.).

1. Sammenfatning og forberedelse af dsRNA
<ol><li>Identificere dsRNA målwebsteder i den 3' utranslaterede region af target gener. DsTub bruges til at målrette α-tubulin (badekar) gen (GenBank stammesamlingsnummer: KX228233), og dsEGFP som en negativ dsRNA kontrol er designet fra sekvensen af forbedret grønne fluorescens protein (EGFP; GenBank stammesamlingsnummer: LC311024).</li>
	<li>Udføre standard PCR-amplifikation for at syntetisere dsRNA skabeloner med gen-specifikke primere indeholdende T7 promotor sekvens (...

<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=Chapter+Five+-+Biological+Mechanisms+Determining+the+Success+of+RNA+Interference+in+Insects.">Chapter Five - Biological Mechanisms Determining the Success of RNA Interference in Insects.</a> International Review of Cell and Molecular Biology. 312, 139-167 (2014).</li><li>San Miguel, K., Scott, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+next+generation+of+insecticides:+dsRNA+is+stable+as+a+foliar-applied+insecticide.">The next generation of insecticides: dsRNA is stable as a foliar-applied insecticide.</a> Pest Manag. Sci. 72, 801-809 (2016).</li><li>Scott, J. G., 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=Towards+the+elements+of+successful+insect+RNAi.">Towards the elements of successful insect RNAi.</a> J. Insect Physiol. 59, 1212-1221 (2013).</li><li>Joga, M. R., Zotti, M. J., Smagghe, G., Christiaens, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi+efficiency,+systemic+properties,+and+novel+delivery+methods+for+pest+insect+control:+What+we+know+so+far.">RNAi efficiency, systemic properties, and novel delivery methods for pest insect control: What we know so far.</a> Front. Physiol. 7, (2016).</li><li>Garbutt, J. S., Bell&#233;s, X., Richards, E. H., Reynolds, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistence+of+double-stranded+RNA+in+insect+hemolymph+as+a+potential+determiner+of+RNA+interference+success:+Evidence+from+Manduca+sexta+and+Blattella+germanica.">Persistence of double-stranded RNA in insect hemolymph as a potential determiner of RNA interference success: Evidence from Manduca sexta and Blattella germanica.</a> J. Insect Physiol. 59, 171-178 (2013).</li><li>Arimatsu, Y., Kotani, E., Sugimura, Y., Furusawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+a+cDNA+encoding+extracellular+dsRNase+and+its+expression+in+the+silkworm,+Bombyx+mori.">Molecular characterization of a cDNA encoding extracellular dsRNase and its expression in the silkworm, Bombyx mori.</a> Insect Biochem. Mol. Biol. 37, 176-183 (2007).</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=Variation+in+RNAi+efficacy+among+insect+species+is+attributable+to+dsRNA+degradation+in+vivo.">Variation in RNAi efficacy among insect species is attributable to dsRNA degradation in vivo.</a> Insect Biochem. Mol. Biol. 77, 1-9 (2016).</li><li>Wynant, 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=Identification,+functional+characterization+and+phylogenetic+analysis+of+double+stranded+RNA+degrading+enzymes+present+in+the+gut+of+the+desert+locust,+Schistocerca+gregaria.">Identification, functional characterization and phylogenetic analysis of double stranded RNA degrading enzymes present in the gut of the desert locust, Schistocerca gregaria.</a> Insect Biochem. Mol. Biol. 46, 1-8 (2014).</li><li>Huang, J. -. H., Belles, X., Lee, H. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+characterization+of+hypertrehalosemic+hormone+receptor+in+relation+to+hemolymph+trehalose+and+to+oxidative+stress+in+the+cockroach+Blattella+germanica.">Functional characterization of hypertrehalosemic hormone receptor in relation to hemolymph trehalose and to oxidative stress in the cockroach Blattella germanica.</a> Exp. Endocrinol. 2, 114 (2012).</li><li>Lin, Y. -. H., Lee, C. -. M., Huang, J. -. H., Lee, H. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+regulation+of+permethrin+susceptibility+by+glutathione+S-transferase+(BgGSTD1)+in+the+German+cockroach+(Blattella+germanica).">Circadian regulation of permethrin susceptibility by glutathione S-transferase (BgGSTD1) in the German cockroach (Blattella germanica).</a> J. Insect Physiol. 65, 45-50 (2014).</li><li>Lozano, J., Kayukawa, T., Shinoda, T., Belles, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+taiman+in+insect+metamorphosis.">A role for taiman in insect metamorphosis.</a> PLOS Genet. 10, 1004769 (2014).</li><li>Lozano, J., Monta&#241;ez, R., Belles, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MiR-2+family+regulates+insect+metamorphosis+by+controlling+the+juvenile+hormone+signaling+pathway.">MiR-2 family regulates insect metamorphosis by controlling the juvenile hormone signaling pathway.</a> Proc. Natl. Acad. Sci. 112, 3740-3745 (2015).</li><li>Lin, Y. -. H., Huang, J. -. H., Liu, Y., Belles, X., Lee, H. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+delivery+of+dsRNA+lipoplexes+to+German+cockroach+protects+dsRNA+from+degradation+and+induces+RNAi+response.">Oral delivery of dsRNA lipoplexes to German cockroach protects dsRNA from degradation and induces RNAi response.</a> Pest Manag. Sci. 73, 960-966 (2017).</li><li>Gharavi, 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=Chiral+cationic+polyamines+for+chiral+microcapsules+and+siRNA+delivery.">Chiral cationic polyamines for chiral microcapsules and siRNA delivery.</a> Bioorg. Med. Chem. Lett. 23, 5919-5922 (2013).</li><li>Whyard, S., Singh, A. D., Wong, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ingested+double-stranded+RNAs+can+act+as+species-specific+insecticides.">Ingested double-stranded RNAs can act as species-specific insecticides.</a> Insect Biochem. Mol. Biol. 39, 824-832 (2009).</li><li>Luo, 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=Differential+responses+of+migratory+locusts+to+systemic+RNA+interference+via+double-stranded+RNA+injection+and+feeding.">Differential responses of migratory locusts to systemic RNA interference via double-stranded RNA injection and feeding.</a> Insect Mol. Biol. 22, 574-583 (2013).</li><li>Liu, J., Smagghe, G., Swevers, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+response+of+BmToll9-1+and+RNAi+machinery+genes+to+exogenous+dsRNA+in+the+midgut+of+Bombyx+mori.">Transcriptional response of BmToll9-1 and RNAi machinery genes to exogenous dsRNA in the midgut of Bombyx mori.</a> J. Insect Physiol. 59, 646-654 (2013).</li><li>Airs, P. M., Bartholomay, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+for+mosquito+and+mosquito-borne+disease+control.">RNA interference for mosquito and mosquito-borne disease control.</a> Insects. 8, 4 (2017).</li><li>Taning, C. N. 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Denne protokol udgør en metode til effektiv RNAi gennem oral levering af dsRNA lipoplexes, der omfatter beskyttelse mod ribonuklease fordøjelsen i midgut juice af tysk kakerlak. Som vist i andre undersøgelser vedrørende forskellige insektarter, regnskabsmæssigt den fattige RNAi virkning gennem oral levering af dsRNA for det meste ved nedbrydningen af dsRNA8,9,10. Denne protokol producerer Liposomer, der tjener som beskytten...

RNAi er blevet bevist som en effektiv metode til knockdown genekspression gennem en mekanisme for en post-transcriptional silencing pathway udløst af dsRNA molekyler i mange eukaryoter1. I det sidste årti af undersøgelse, er RNAi blevet et nyttigt redskab til at studere funktionerne af gener fra udvikling til opførsel af nedbryder udtryk for specifikke gener via injektion og/eller fodring af dsRNA i forskellige taxa af insekter2,3. Specificitet og robusthed af de ozonlagsnedbrydende virkning, er anvendelsen af RNAi i øjeblikket betragtes som en potentiel strategi for pest control management4,5. Dog, effektiviteten af RNAi varierer meget mellem insektarter, afhængigt af de forskellige gener bliver målrettet og leveringsmetoder. En voksende mængde af beviser tyder på, at ustabilitet i dsRNA, som er nedbrudt af ribonukleaser, er en kritisk faktor i en begrænset effekt af RNAi5,6. For eksempel, er lave RNAi følsomhed i Manduca sexta blevet forklaret ved, at dsRNA blandet med hemolymph var hurtigt forringede senest 1 time7. På samme måde, tilstedeværelsen af alkalisk nukleaser i midgut, som effektivt forringe indtaget dsRNA, er stærkt korreleret med lav RNAi effektivitet i forskellige insekt ordrer8,9,10.
Oral levering af dsRNA er særligt interessant for anvendelsen af RNAi i en strategi til kontrol med pest, men en metode til at forsinke nedbrydning af dsRNA af nukleaser i midgut har ikke endnu blevet udviklet, som ville have potentiale til at sikre en effektiv RNAi gennem fodring. Dog er passivitet af RNAi til oral levering af dsRNA blevet rapporteret af fodring stor mængde dsRNA, fx 50 µg i Bombyx mori, eller konstant fodring i 8 dage (8 µg dsRNA i alt) i arternes locust. Den tyske kakerlak, Blattella germinica, er meget følsom over for RNAi ved injektion af dsRNA11,12,13,14, men er ikke lydhør over for dsRNA gennem fodring. For nylig, Lin et al. (2017) har vist, at dsRNA indkapslet med Liposom luftfartsselskaber resultater i vellykket RNAi til knockdown α-tubulin genekspression i den midgut og udløser betydelig dødelighed af tysk kakerlak15. Som nedbrydning af dsRNA i midgut er den begrænsende faktor for mundtlige RNAi, tjene Liposom luftfartsselskaber som et middel til at beskytte dsRNA mod nedbrydning, som er umiddelbart gældende i andre insekter med stærk nukleasen aktiviteter i tarmen. Af note, grunden til at vælge bestemte Transfektion reagens (Se Tabel af materialer) vi brugt som Liposom luftfartsselskab i den nuværende protokol er at det er blevet testet for insekt celle linje Transfektion med mindre toksicitet, ifølge den producentens anvisninger. I henhold til sammenligning af forskellige Liposom Transfektion systemer i Gharavi et al. (2013) 16, effektiviteten af transfecting lille interfererende RNA (siRNA) er omtrent den samme mellem denne og andre kommercielt tilgængelige systemer, der har været brugt i dsRNA levering systemer i andre insekter17,18 . Desuden er vores fodring metode forsigtig nok til at sikre den rette mængde af dsRNA indtages af hver kakerlak, og at resultaterne er robust og bekræftet. I Resumé viser denne protokol og resultater, at bruge dsRNA lipoplexes forbedrer dsRNA stabilitet og åbner døren til udformningen af strategien oral levering af RNAi, som er en lovende tilgang til skadedyrsbekæmpelse i fremtiden.

<table border="0" cellpadding="0" cellspacing="0" style="width:1124px;" width="1123">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">GenJe Plus DNA in vitro Transfection reagent</td>
			<td style="width:133px;">SignaGen</td>
			<td style="width:157px;">SL100499</td>
			<td style="width:481px;">for lipoplexes preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blend Taq plus</td>
			<td style="width:133px;">TOYOBO&#160;</td>
			<td style="width:157px;">BTQ-201</td>
			<td>for PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fast SYBR Green Master Mix</td>
			<td style="width:133px;">ABI&#160;</td>
			<td style="width:157px;">4385612</td>
			<td>for qPCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">FirstChoice RLM-RACE Kit</td>
			<td style="width:133px;">Invitrogen</td>
			<td style="width:157px;">AM1700</td>
			<td>for 3&#39; UTR identification&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">MEGAscript T7 Transcription Kit</td>
			<td style="width:133px;">Invitrogen</td>
			<td style="width:157px;">AMB13345</td>
			<td>for dsRNA synthesis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TURBO DNase</td>
			<td style="width:133px;">Invitrogen</td>
			<td style="width:157px;">AM2239</td>
			<td>remove DNA template from dsRNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TRIzol</td>
			<td style="width:133px;">Invitrogen</td>
			<td style="width:157px;">15596018</td>
			<td>for dsRNA or total RNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RQ1 RNase-Free Dnase</td>
			<td style="width:133px;">Promega</td>
			<td style="width:157px;">M6101</td>
			<td>remove DNA template from total RNA&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">chloroform&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:157px;">C2432</td>
			<td>for dsRNA or total RNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">2-Propanol</td>
			<td>Sigma-Aldrich</td>
			<td style="width:157px;">I9516</td>
			<td>for dsRNA or total RNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>24102</td>
			<td>for dsRNA or total RNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Diethyl pyrocarbonate, DEPC</td>
			<td>Sigma-Aldrich</td>
			<td style="width:157px;">D5758</td>
			<td>for RNase free water preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">glucose solution</td>
			<td>Sigma-Aldrich</td>
			<td style="width:157px;">G3285</td>
			<td>for lipoplexes preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride, NaCl</td>
			<td>Sigma-Aldrich</td>
			<td style="width:157px;">S7653</td>
			<td>insect saline buffer formula</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Potassium chloride, KCl</td>
			<td>Sigma-Aldrich</td>
			<td style="width:157px;">P9333</td>
			<td>insect saline buffer formula</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:352px;">Calcium chloride, CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C1016</td>
			<td>insect saline buffer formula</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Magnesium chloride hexahydrate, MgCl2.6H2O</td>
			<td>Sigma-Aldrich</td>
			<td style="width:157px;">M2670</td>
			<td>insect saline buffer formula</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">EGTA&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:157px;">E3889</td>
			<td>enzyme inhibitor&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dissecting scissor</td>
			<td style="width:133px;">F.S.T.</td>
			<td style="width:157px;"></td>
			<td>cockroach dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fine tweezers</td>
			<td style="width:133px;">F.S.T.</td>
			<td style="width:157px;"></td>
			<td>cockroach dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">flexible tweezer</td>
			<td style="width:133px;">F.S.T.</td>
			<td style="width:157px;"></td>
			<td>cockroach holding&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pipetman</td>
			<td style="width:133px;">RAININ</td>
			<td style="width:157px;">P10</td>
			<td>sample preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">microcentrifuge tube</td>
			<td style="width:133px;">Axygen</td>
			<td>MCT175C, PCR02C</td>
			<td>sample preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">pipette tip&#160;</td>
			<td style="width:133px;">Axygen</td>
			<td></td>
			<td>sample preparation</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">vortexter</td>
			<td style="width:133px;">Digisystem</td>
			<td style="width:157px;">vm1000</td>
			<td>sample preparation</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Minispin centrifuge</td>
			<td style="width:133px;">The Gruffin Group</td>
			<td style="width:157px;">GMC 206</td>
			<td>for liquid spin down&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td style="width:133px;">ALC</td>
			<td style="width:157px;">PK121R</td>
			<td>sample preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pH meter&#160;</td>
			<td style="width:133px;">JENCO</td>
			<td style="width:157px;">6071</td>
			<td>for pH adjust</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">micro-volume spectrophotometer</td>
			<td style="width:133px;">Quawell</td>
			<td style="width:157px;">Q3000</td>
			<td>nucleic acid quantitative</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">PCR Thermal cycler</td>
			<td style="width:133px;">ABI&#160;</td>
			<td style="width:157px;">2720</td>
			<td>for template PCR or&#160; dsRNA synthesis incubation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">quantitative real-time PCR</td>
			<td style="width:133px;">ABI&#160;</td>
			<td style="width:157px;">StepOne plus</td>
			<td>gene expression quantitative</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifugal Vacuum Concentrators</td>
			<td style="width:133px;">eppendorf</td>
			<td style="width:157px;">5301</td>
			<td>for dsRNA or total RNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multipette&#160;</td>
			<td style="width:133px;">eppendorf</td>
			<td style="width:157px;">xstream</td>
			<td>for real-time PCR sample loading&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose I</td>
			<td style="width:133px;">amresco</td>
			<td style="width:157px;">0710</td>
			<td>for nucleic acid electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">tub gene specfifc forward preimer</td>
			<td style="width:133px;">tri-I biotech</td>
			<td style="width:157px;"></td>
			<td>GGG ACA AGC CGG AGT GCA GA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">tub gene specfifc reverse preimer</td>
			<td style="width:133px;">tri-I biotech</td>
			<td style="width:157px;"></td>
			<td>TCC TGC TCC TGT CTC GCT GA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">dsTub template forward primer</td>
			<td style="width:133px;">tri-I biotech</td>
			<td style="width:157px;"></td>
			<td>TAA TAC GAC TCA CTA TAG GGA CAA GCC GGA GTG CAG&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">dsTub template reverse primer</td>
			<td style="width:133px;">tri-I biotech</td>
			<td style="width:157px;"></td>
			<td>TAA TAC GAC TCA CTA TAG GGT CCT GCT CCT GTC TCG CTG&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">dsEGFP template forward preimer</td>
			<td style="width:133px;">tri-I biotech</td>
			<td style="width:157px;"></td>
			<td>TAA TAC GAC TCA CTA TAG GGT ATG GTG AGC AAG GGC GAG GAG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">dsEGFP template reverse preimer</td>
			<td style="width:133px;">tri-I biotech</td>
			<td style="width:157px;"></td>
			<td>TAA TAC GAC TCA CTA TAG GGT GGC GGA TCT TGA AGT TCA CC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">tub qPCR forward primer</td>
			<td style="width:133px;">tri-I biotech</td>
			<td style="width:157px;"></td>
			<td>GGA CCG CAT CAG GAA ACT GGC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">tub qPCR reverse preimer</td>
			<td style="width:133px;">tri-I biotech</td>
			<td style="width:157px;"></td>
			<td>CCA CAG ACA GCC TCT CCA TGA GC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">ef1 qPCR forward primer</td>
			<td style="width:133px;">tri-I biotech</td>
			<td style="width:157px;"></td>
			<td>CGC TTG AGG AAA TCA AGA AGG A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">ef1 qPCRreverse preimer</td>
			<td style="width:133px;">tri-I biotech</td>
			<td style="width:157px;"></td>
			<td>CCT GCA GAG GAA GAC GAA G</td>
		</tr>
	</tbody>
</table>

practical use of rna interference: oral delivery of double-stranded rna in liposome carriers for cockroaches

RNA-interferens (RNAi) er blevet bredt anvendt til afdække de biologiske funktioner af mange gener, og har været tænkt som en pest kontrol værktøj fungerer ved afbrydelse af væsentlige genekspression. Selv om forskellige metoder, såsom injektion, fodring, og iblødsætning, er blevet rapporteret om succesfulde levering af dobbelt-strenget RNA (dsRNA), er RNAi effektivitet gennem oral levering af dsRNA meget varierende blandt forskellige insekt grupper. Den tyske kakerlak, Blattella germanica, er meget følsom over for injektion af dsRNA, som det fremgår af mange undersøgelser offentliggjort tidligere. Den nuværende undersøgelse beskriver en metode til at påvise, at dsRNA indkapslet med Liposom luftfartsselskaber er tilstrækkelig til at forsinke nedbrydning af dsRNA af midgut juice. Især, kontinuerlig fodring af dsRNA indkapslet af Liposomer betydeligt reducerer tubulin udtryk i midgut, og førte til døden af kakerlakker. Afslutningsvis, kunne formulering og anvendelse af dsRNA lipoplexes, som beskytter dsRNA mod nukleaser, være en praktisk anvendelse af RNAi for insekt Skadedyrsbekæmpelse i fremtiden.

En forenklet ordning i protokollen for den mundtlige levering af dsRNA er præsenteret i figur 1, hvor de vigtigste skridt til forberedelse af dsRNA lipoplexes er vist.
For at undersøge den beskyttelse af Liposom luftfartsselskaber ved dsRNA nedbrydning i midgut juice af B. germanica, en ex vivo assay hvor dsTub lipoplexes blev inkuberet med midgut juice blev gennemført og integr...

Watch this Scientific Journal Video about Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches at JoVE.com

Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches

Praktisk brug af RNA-interferens: Oral levering af dobbelt-strenget RNA i Liposom bærere af kakerlakker

Dette manuskript viser nedbrydningen af genekspression i midgut af tysk kakerlak gennem oral indtagelse af dobbelt-strenget RNA indkapslet i Liposomer.

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool ...

Research

JoVE Journal

Biology

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro Transskription og lukning af Gaussia luciferase-mRNA Efterfulgt af HeLa Celletransfektion

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Chromatin Isolation by RNA Purification (ChIRP)

Chromatin Isolation by RNA Purification ChIRP

Chromatin Isolering af RNA Oprensning (chirp)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Substrate Generation for Endonucleases of CRISPR/Cas Systems

Substrat Generation for endonukleaser af CRISPR / Cas Systems

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Brug af RNA-medieret Interferens fodringsstrategien at screene for gener involveret i Body Size forordning i Nematode<em&gt; C. elegans</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq

Udtømning af ribosomalt RNA for Mosquito Gut Metagenomic RNA-seq

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity ...

RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes

RNA Catalyst som en reporter for screening af lægemidler mod RNA redigering i trypanosomer

Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been ...

Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum

Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum

Larve RNA-interferens i Den Røde rismelbille,<em&gt; Tribolium castaneum</em

The red flour beetle, Tribolium&#160;castaneum, offers a repertoire of experimental tools for genetic and developmental studies, including a fully ...

Production of Double-stranded DNA Ministrings

Produktion af Double DNA Ministrings

We constructed linear covalently closed (LCC) DNA minivectors as a non-viral gene-delivery vector alternative produced via a simple platform in vivo. DNA ...

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

TChIP-Seq: Celle-Type-specifikke Epigenome profilering

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...