Funding from the Kansas Agricultural Experiment Station and K-State Arthropod Genomics Center to KYZ supported the original development of chitosan/dsRNA-mediated gene silencing in A. gambiae19.The A. gambiae work described here was supported in part by R01-AI095842 from NIH/NIAID to KM. Development of chitosan/siRNA-mediated gene silencing in A. aegypti20,21 was supported by NIH/NIAID Award R01-AI081795 to MDS. The contents of this study are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

1. Mosquito Specie e allevamento <ol><li> Mantenere A. gambiae G3 e A. ceppi aegypti Liverpool IB12 (utilizzati negli studi rappresentativi sotto) o da altri ceppi di interesse secondo prassi di laboratorio standard o come descritto in precedenza 23,24. </li></ol> 2. dsRNA e siRNA Progettazione e Produzione <ol><li> Primer design per costruire modelli lunghi dsRNA specifiche per il gene di interesse. Utilizzare lo strumento E-RNAi 25. Produrre dsRNA secondo il metodo di scelta o come descritto in precedenza 19. <ol><li> Per un controllo negativo, sintetizzare dsRNA 19 corrispondente alla sequenza della proteina fluorescente verde (GFP), β-galattosidasi (β-gal), o qualche altro gene non espresso in zanzare. Se lo si desidera, dsRNA 19 utilizzato per generare i risultati rappresentativi riassunte di seguito può essere utilizzato come controllo positivo. Conservare dsRNA disciolto in acqua priva di RNAse a -80 °; C fino al momento dell&#39;uso. </li></ol></li><li> In alternativa, il design siRNA specifici gene secondo la prassi di laboratorio standard o come descritto in precedenza 10. Rimescolare la sequenza di un knockdown siRNA progettare siRNA controllo negativo che non corrisponde a qualsiasi gene zanzara. Acquistare i siRNA personalizzati, disponibili attraverso una serie di fornitori affidabili. <ol><li> Se lo si desidera, siRNA 20,21 utilizzato per ottenere i risultati rappresentativi riassunte di seguito possono essere utilizzati come controlli positivi. Conservare siRNA disciolto in acqua RNase-free a -80 ° C fino al momento. </li></ol></li></ol> 3. Preparare chitosano / RNA interferenti nanoparticelle <ol><li> Raccogliere RT 100 mM sodio solfato pre-made (100 mM Na 2 SO 4 in H 2 O deionizzata) e 0,1 M di acetato di sodio (0,1 M NaC 2 H 3 O 2 -0.1 M acido acetico, pH 4,5 in deionizzata H 2 O) buffer. </li><li> Sciogliere chitosano (≥75%deacetilato) in tampone acetato 0,1 M di sodio per rendere 0,02% (w / v) Soluzione di lavoro e mantenere la soluzione a temperatura ambiente prima dell&#39;uso. </li><li> Sciogliere dsRNA o siRNA in 50 ml di acqua deionizzata H 2 O e aggiungere al buffer solfato di sodio 100 mM per ottenere una soluzione a 100 ml di 32 mg di dsRNA / siRNA per 100 ml di 50 mM solfato di sodio. </li><li> Aggiungere 100 pl di soluzione di chitosano alla soluzione dsRNA / siRNA e poi riscaldare la miscela in un bagno di acqua a 55 ° C per 1 min. Impostare un controllo aggiungendo 100 ml di 50 mM sodio solfato a 100 ml di soluzione di chitosano e seguire la stessa procedura. </li><li> Mescolare le soluzioni immediatamente per 30 sec nel vortex ad alta velocità a RT per facilitare la formazione di nanoparticelle. </li><li> Centrifugare la miscela a 13.000 xg per 10 min a RT, dopo di che una pastiglia deve essere visibile. Trasferire il surnatante in una nuova provetta da 1,5 ml. Asciugare il pellet per ≈10 min a RT prima di utilizzarla per preparare il cibo zanzare. </li><li> Misura la concentrazione di dsRNA / siRNA nel surnatante utilizzando il surnatante dal controllo (cfr 3,5) come uno spazio vuoto per calcolare la quantità totale di dsRNA / siRNA che è rimasto nel surnatante. Utilizzare la differenza tra gli importi di base delle dsRNA / siRNA e gli importi residui nei sopranatanti per calcolare la percentuale di dsRNA / siRNA intrappolato in nanoparticelle. Questa efficienza di caricamento è normalmente superiore al 90%. </li><li> Ripetere la stessa procedura se sono necessari ulteriori nanoparticelle. Utilizzare immediatamente le nanoparticelle secche. L&#39;impatto di conservazione frigorifera di particelle prima dell&#39;uso non è stata valutata. </li></ol> 4. Preparazione Mosquito alimentare contenente chitosano / RNA interferenti nanoparticelle <ol><li> Preparare 1 ml di una soluzione di agarosio al 2% (w / v) in acqua deionizzata, sciogliere l&#39;agarosio, e conservare la soluzione di agarosio fuso in un bagno di acqua 55 C prima dell&#39;uso. </li><li> Mescolare fiocchi alimentari di pesce (47% di proteine ​​gregge, min. 10% grassi greggi, max. Fibra grezza 3%) e lievito secco in unrapporto di 2: 1 (w / w). Macinare il composto a piccole particelle con un mortaio e pestello (percorribile attraverso n ° 50 USA setaccio di prova standard). Utilizzare il cibo a terra, che dovrebbe essere di colore marrone, immediatamente o conservarla in un contenitore sigillato a 4 ° C per diverse settimane. </li><li> In una provetta da 1,5 ml, mescolare 6 mg di cibo a terra con le nanoparticelle secche Sezione 3.7 con uno stuzzicadenti. </li><li> Aggiungere 30 ml di soluzione di gel di agarosio 2% pre-fusa alla miscela alimentare nanoparticelle; mescolate immediatamente e accuratamente utilizzando una punta stuzzicadenti o pipetta. </li><li> Utilizzare il gel contenente cibo e nanoparticelle per alimentare immediatamente le larve di zanzara. In alternativa, una volta che il gel è completamente solidificato a temperatura ambiente, memorizzare il gel a 4 ° C e utilizzare il giorno successivo, oa -80 ° C per un uso successivo. </li></ol> 5. Alimentazione Mosquito Larve con alimenti contenenti chitosano / RNA interferenti nanoparticelle <ol><li> Alimentazione A. gambiae larve di zanzara: <ol><li> Rimuovere un single gel pellet dal tubo di 1,5 ml con uno stuzzicadenti e tagliarlo a 6 fette uguali con una lama di rasoio pulito o stuzzicadenti. </li><li> Trasferimento 20 larve al terzo stadio di una piastra di Petri da 500 ml contenente 100 ml di acqua deionizzata. </li><li> Feed le larve della zanzara aggiungendo una fetta di pellet gel (tritato in pezzi più piccoli) a piatto Petri una volta al giorno per quattro giorni. Assicurarsi di osservare l&#39;alimentazione delle larve sul pellet, che dovrebbe essere ridotto significativamente in dimensioni o del tutto assente dal giorno successivo. Dopo il tempo, le zanzare si svilupperà in fine del quarto larve instar. </li><li> Registrare eventuali modifiche fenotipiche visibili durante l&#39;esperimento. Esaminate i livelli di trascrizione e altre variazioni fenotipiche come discusso nella sezione 6 alla fine del periodo di quattro giorni. </li></ol></li><li> Alimentazione A. aegypti larve di zanzara: <ol><li> Tagliare il pellet di gel in 6 fette uguali con una lama di rasoio pulito o stuzzicadenti. </li><li> Posizionare i 50 anni-sincronizzato 24 ore dopo la schiusa delle uova fiprima instar larve in una capsula di Petri in ≈40 ml di acqua deionizzata. </li><li> Larve si nutrono una fetta per ogni capsula di Petri per 4 ore / giorno, quindi trasferire le larve di nuovo alla dieta larvale regolare di 2: 1 a terra fiocchi alimentari di pesce e di lievito secco per il resto della giornata. Ripetere quotidianamente nei quattro stadi larvali. </li><li> Esaminare i livelli di trascrizione e di altri cambiamenti fenotipici come discusso nella sezione 6 nei punti di tempo di sviluppo desiderati. </li></ol></li></ol> 6. Conferma di Gene Knockdown <ol><li> Relativa trascrizione quantificazione da qRT-PCR in A. aegypti e A. gambiae larve. <ol><li> Eseguire ed analizzare saggi qRT-PCR con tre repliche biologiche su almeno il 10 pool di controllo contro le larve sperimentale riportato 11,19,20, o secondo procedure di laboratorio standard. I risultati rappresentativi sono stati inseriti. </li><li> Per l&#39;analisi di una parte particolare tipo di tessuto / corpo (es., Il cervello o antenna), PERFOrm qRT-PCR come descritto nella seguente 6.1.1 dissezione per recuperare il tessuto di interesse (ad esempio, vedere Mysore et al. 21). </li></ol></li><li> Conferma della atterramento da ibridazione in situ <ol><li> Sintetizzare antisenso digossigenina marcato e percepire riboprobes controllo secondo le prassi di laboratorio standard o come descritto 26. </li><li> Eseguire ibridazione in situ per il protocollo Haugen et al. 27 per la conferma di atterramento come descritto in precedenza 11,20 e discusso nella sezione risultati rappresentativi di seguito. </li></ol></li><li> La conferma di atterramento immunoistochimica <ol><li> Se sono disponibili anticorpi contro il prodotto proteico del gene targeting, eseguire immunoistochimica come descritto in precedenza 28, che facilita l&#39;identificazione dei soggetti con i maggiori livelli di knockdown per l&#39;analisi del fenotipo 20 come esemplificato nella representative la sezione risultati qui sotto. </li></ol></li></ol>

<ol>
	<li>Knipling, E. F., 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=Genetic+control+of+insects+of+public+health+importance.">Genetic control of insects of public health importance.</a> Bulletin of the World Health Organization. 38 (3), 421-438 (1968).</li><li>Fu, 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=Female-specific+flightless+phenotype+for+mosquito+control.">Female-specific flightless phenotype for mosquito control.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (10), 4550-4554 (2010).</li><li>Wise de Valdez, M. R., 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=Genetic+elimination+of+dengue+vector+mosquitoes.">Genetic elimination of dengue vector mosquitoes.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (12), 4772-4775 (2011).</li><li>Harris, A. F., 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=Successful+suppression+of+a+field+mosquito+population+by+sustained+release+of+engineered+male+mosquitoes.">Successful suppression of a field mosquito population by sustained release of engineered male mosquitoes.</a> Nature Biotechnology. 30 (9), 828-830 (2012).</li><li>Fire, A., 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=Potent+and+specific+genetic+interference+by+double-stranded+RNA+in+Caenorhabditis+elegans.">Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.</a> Nature. 391 (6669), 806-811 (1998).</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 science. 20 (1), 4-14 (2013).</li><li>Zhang, H., Li, H. C., Feasibility Miao, X. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=limitation+and+possible+solutions+of+RNAi-based+technology+for+insect+pest+control.">limitation and possible solutions of RNAi-based technology for insect pest control.</a> Insect science. 20 (1), 15-30 (2013).</li><li>Clemons, A., Haugen, M., Severson, D. W., Duman-Scheel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+genes+in+Aedes+aegypti+embryos.">Functional analysis of genes in Aedes aegypti embryos.</a> Cold Spring Harb Protoc. 2010 (10), (2010).</li><li>Clemons, A., 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=siRNA-mediated+gene+targeting+in+Aedes+aegypti+embryos+reveals+that+frazzled+regulates+vector+mosquito+CNS+development.">siRNA-mediated gene targeting in Aedes aegypti embryos reveals that frazzled regulates vector mosquito CNS development.</a> PLoS One. 6 (1), e16730 (2011).</li><li>Haugen, M., 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=Semaphorin-1a+is+required+for+Aedes+aegypti+embryonic+nerve+cord+development.">Semaphorin-1a is required for Aedes aegypti embryonic nerve cord development.</a> PLoS One. 6 (6), e21694 (2011).</li><li>Nguyen, C., 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=Functional+genetic+characterization+of+salivary+gland+development+in+Aedes+aegypti.">Functional genetic characterization of salivary gland development in Aedes aegypti.</a> EvoDevo. 4 (1), 9 (2013).</li><li>Sarro, 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=Requirement+for+commissureless2+function+during+dipteran+insect+nerve+cord+development.">Requirement for commissureless2 function during dipteran insect nerve cord development.</a> Developmental dynamics: an official publication of the American Association of Anatomists. 242 (12), 1466-1477 (2013).</li><li>Singh, A. D., Wong, S., Ryan, C. P., Whyard, S. <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+double-stranded+RNA+in+larvae+of+the+yellow+fever+mosquito,+Aedes+aegypti:+implications+for+pest+mosquito+control.">Oral delivery of double-stranded RNA in larvae of the yellow fever mosquito, Aedes aegypti: implications for pest mosquito control.</a> J Insect Sci. 13, 69 (2013).</li><li>Lopez-Martinez, G., Meuti, M., Denlinger, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rehydration+driven+RNAi:+a+novel+approach+for+effectively+delivering+dsRNA+to+mosquito+larvae.">Rehydration driven RNAi: a novel approach for effectively delivering dsRNA to mosquito larvae.</a> Journal of medical entomology. 49 (1), 215-218 (2012).</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> Journal of insect physiology. 59 (12), 1212-1221 (2013).</li><li>Coy, M. R., 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=Gene+silencing+in+adult+Aedes+aegypti+mosquitoes+through+oral+delivery+of+double-stranded+RNA.">Gene silencing in adult Aedes aegypti mosquitoes through oral delivery of double-stranded RNA.</a> J Appl Entomol. 136 (10), 741-748 (2012).</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 molecular biology. 19 (5), 683-693 (2010).</li><li>Mysore, K., Flannery, E. M., Tomchaney, M., Severson, D. W., Duman-Scheel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+Aedes+aegypti+olfactory+system+development+through+chitosan/siRNA+nanoparticle+targeting+of+semaphorin-1a.">Disruption of Aedes aegypti olfactory system development through chitosan/siRNA nanoparticle targeting of semaphorin-1a.</a> PLoS neglected tropical diseases. 7 (5), e2215 (2013).</li><li>Mysore, K., Andrews, E., Li, P., Duman-Scheel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitosan/siRNA+nanoparticle+targeting+demonstrates+a+requirement+for+single-minded+during+larval+and+pupal+olfactory+system+development+of+the+vector+mosquito+Aedes+aegypti.">Chitosan/siRNA nanoparticle targeting demonstrates a requirement for single-minded during larval and pupal olfactory system development of the vector mosquito Aedes aegypti.</a> BMC developmental biology. 14 (1), 9 (2014).</li><li>Dass, C. 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=Chitosan-mediated+orally+delivered+nucleic+acids:+a+gutful+of+gene+therapy.">Chitosan-mediated orally delivered nucleic acids: a gutful of gene therapy.</a> Journal of drug targeting. 16 (4), 257-261 (2008).</li><li>An, C., Budd, A., Kanost, M. R., Michel, K. <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+a+regulatory+unit+that+controls+melanization+and+affects+longevity+of+mosquitoes.">Characterization of a regulatory unit that controls melanization and affects longevity of mosquitoes.</a> Cellular and molecular life sciences : CMLS. 68 (11), 1929-1939 (2011).</li><li>Clemons, A., Mori, A., Haugen, M., Severson, D. W., Duman-Scheel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+and+egg+collection+of+Aedes+aegypti.">Culturing and egg collection of Aedes aegypti.</a> Cold Spring Harb Protoc. 2010 (10), (2010).</li><li>Horn, T., Boutros, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E-RNAi:+a+web+application+for+the+multi-species+design+of+RNAi+reagents--2010+update.">E-RNAi: a web application for the multi-species design of RNAi reagents--2010 update.</a> Nucleic acids research. 38, W332-W339 (2010).</li><li>Patel, N. H., PA, K. r. i. e. g. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+19.+In+situ+hybridization+to+whole+mount+Drosophila+embryos.">Ch. 19. In situ hybridization to whole mount Drosophila embryos.</a> A laboratory guide to RNA: Isolation, Analysis, and Synthesis. , (1996).</li><li>Haugen, M., 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=.">.</a> Whole-mount in situ hybridization for analysis of gene expression during Aedes aegypti development. 2010 (10), (2010).</li><li>Clemons, A., Flannery, E., Kast, K., Severson, D. W., Duman-Scheel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+analysis+of+protein+expression+during+Aedes+aegypti+development.">Immunohistochemical analysis of protein expression during Aedes aegypti development.</a> Cold Spring Harb Protoc. 2010 (10), (1101).</li><li>Rund, S. S., Hou, T. Y., Ward, S. M., Collins, F. H., Duffield, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+profiling+of+diel+and+circadian+gene+expression+in+the+malaria+vector+Anopheles+gambiae.">Genome-wide profiling of diel and circadian gene expression in the malaria vector Anopheles gambiae.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (32), E421-E430 (2011).</li><li>Gaj, T., Gersbach, C. A., Barbas, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ZFN,+TALEN,+CRISPR/Cas-based+methods+for+genome+engineering.">ZFN, TALEN, CRISPR/Cas-based methods for genome engineering.</a> Trends in biotechnology. 31 (7), 397-405 (2013).</li><li>Aryan, A., Myles, K. M., Adelman, Z. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+genome+editing+in+Aedes+aegypti+using+TALENs.">Targeted genome editing in Aedes aegypti using TALENs.</a> Methods. 69 (1), 38-45 (2014).</li><li>Sarathi, M., Simon, M. C., Venkatesan, C., Hameed, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+administration+of+bacterially+expressed+VP28dsRNA+to+protect+Penaeus+monodon+from+white+spot+syndrome+virus.">Oral administration of bacterially expressed VP28dsRNA to protect Penaeus monodon from white spot syndrome virus.</a> Mar Biotechnol (NY). 10 (3), 242-249 (2008).</li><li>He, B., 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=Fluorescent+nanoparticle+delivered+dsRNA+toward+genetic+control+of+insect+pests).">Fluorescent nanoparticle delivered dsRNA toward genetic control of insect pests).</a> Adv Mater. 25 (33), 4580-4584 (2013).</li><li>Giljohann, D. A., Seferos, D. S., Prigodich, A. E., Patel, P. C., Mirkin, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+regulation+with+polyvalent+siRNA-nanoparticle+conjugates.">Gene regulation with polyvalent siRNA-nanoparticle conjugates.</a> Journal of the American Chemical Society. 131 (6), 2072-2073 (2009).</li><li>Davis, M. E., 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=Evidence+of+RNAi+in+humans+from+systemically+administered+siRNA+via+targeted+nanoparticles.">Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles.</a> Nature. 464 (7921), 1067-1070 (2010).</li><li>Molinaro, R., 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=Polyethylenimine+and+chitosan+carriers+for+the+delivery+of+RNA+interference+effectors.">Polyethylenimine and chitosan carriers for the delivery of RNA interference effectors.</a> Expert opinion on drug delivery. 10 (12), 1653-1668 (2013).</li><li>Singha, 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=Polymers+in+small-interfering+RNA+delivery.">Polymers in small-interfering RNA delivery.</a> Nucleic acid therapeutics. 21 (3), 133-148 (2011).</li></ol>

The authors have nothing to disclose.

Il chitosano / interferenti metodologia nanoparticelle RNA qui descritto è stato utilizzato per indirizzare efficacemente i geni durante lo sviluppo larvale in A. gambiae (figure 2, 3) e A. aegypti (figure 4, 5, 6, Tabelle 1, 2). Nanoparticelle chitosano possono essere preparati sia con lunghi dsRNA o siRNA, entrambi i quali sono stati utilizzati con successo in zanzare, come dimostrano i risultati rappresentativi qui descritti. Sintesi di dsRNA è meno costoso rispet...

Zanzare sanguigni che alimentano vettore dei generi anofele e Aedine trasmettere agenti patogeni responsabili di alcuni dei peggiori flagelli dell&#39;umanità. Si stima che 3,4 miliardi di persone sono a rischio di contrarre la malaria, che è responsabile di oltre mezzo milione di morti ogni anno in tutto il mondo. Risultati malaria da infezione da Plasmodium sp. Parassiti, che sono trasmessi alle persone attraverso le punture di zanzare infette del genere Anopheles, tra cui il principale vettore africano Anopheles gambiae ( <a href="http://www.who.int/topics/malaria/en/">http://www.who.int/topics/malaria/en/</a> , 2014) 1. Aedes aegypti è la zanzara vettore primario per il virus dengue, che causa la febbre dengue, una malattia febbrile aspecifica che è la più diffusa e significativa malattia arbovirali nel mondo. Virus dengue è attualmente una minaccia per la&gt; 2,5 miliardi di persone ai tropici, con un inciden annualece di circa 50 milioni di casi con conseguente ~ 24.000 decessi all&#39;anno ( <a href="http://www.cdc.gov/dengue/">http://www.cdc.gov/dengue/</a> , 2014) 2. Nonostante l&#39;impatto globale devastante di malattie trasmesse dalle zanzare sulla salute umana, mezzo efficace per prevenire e curare queste malattie sono carenti. Controllo delle zanzare è attualmente il miglior metodo di prevenzione delle malattie. Il potenziale per il controllo delle malattie trasmesse da artropodi di manipolazione genetica degli insetti vettori è stato riconosciuto da più di quattro decenni 3. Ceppi transgenici di A. aegypti progettato per avere un reprimibile specifico femminile di volare fenotipo hanno recentemente fatto il potenziale per l&#39;utilizzo di strategie di controllo vettoriale transgenici una realtà 4-6. Questi progressi hanno sfidato i ricercatori di individuare bersagli genetici nuovi per il controllo vettoriale e ulteriori mezzi di manipolare la funzione del gene in zanzare vettore. L&#39;alterazione di espressione genica durante sviluppo, come è avvenuto in zanzare femmina-volare 4, può favorire il chiarimento delle nuove strategie di controllo vettoriale. Tuttavia, soprattutto a causa sfide tecniche, le funzioni di pochissimi geni sono stati caratterizzati durante lo sviluppo di A. gambiae, A. aegypti, o altre specie di zanzare. Fin dalla sua scoperta nel C. elegans 7, interferenza RNA (RNAi), che viene conservata in animali, piante e microorganismi, è stato ampiamente utilizzato per studi genetici funzionali in un&#39;ampia varietà di organismi, compresi gli insetti 8,9. Il percorso RNAi è iniziata da Dicer, che scinde lungo dsRNA in brevi 20-25 siRNA nucleotidi a lungo che funzionano come specifica sequenza RNA interferenti. geni silenzio siRNA complementari in sequenza attraverso la promozione di fatturato trascrizione, scissione, e la rottura di traduzione 9. Molecole dsRNA lunghi (in genere 300-600 bp) o siRNA targeting personalizzato una particulasequenza r può essere utilizzato in laboratorio ricerca di silenziamento qualsiasi gene di interesse. Gestendo quando interfering RNA viene consegnato, i ricercatori possono controllare il momento in cui silenziamento genico iniziati. Questo vantaggio è utile in quanto può essere utilizzato per superare sfide come letalità sviluppo o sterilità, che possono ostacolare la produzione e manutenzione di ceppi recanti mutazioni ereditarie, un processo costoso e laborioso che non è ancora disponibile in tutte le specie di insetti. Benché il grado di silenziamento genico da RNAi può variare da gene gene, tessuto a tessuto e animale all&#39;altro, RNAi è ampiamente usato per analisi funzionale di geni in zanzare ed altri insetti 8,9. Tre interferenti strategie consegna RNA sono state utilizzate in zanzare: microiniezione, ammollo / applicazione topica, e l&#39;ingestione. Per una storia dettagliata e confronto tra l&#39;utilizzo di queste tre tecniche di insetti, consultare Yu et al 8. We hanno utilizzato con successo la microiniezione 10 come un mezzo per trasmettere siRNA per indirizzare geni dello sviluppo in A. embrioni aegypti, larve, pupe e 11-14. Tuttavia, questa strategia consegna intensità di lavoro richiede sia una configurazione microiniezione e una mano esperta. Inoltre, microiniezione è stressante per l&#39;organismo, un fattore di confusione, in particolare quando saranno valutati fenotipi comportamentali. Infine, la consegna microiniezione non può essere esteso al campo per il controllo vettoriale. In alternativa, l&#39;organismo ammollo in soluzione RNA interferente è anche diventare un mezzo popolare di indurre silenziamento genico, come è conveniente e richiede attrezzature poco o di lavoro. Ammollo è stata applicata principalmente nella linea cellulare di insetto studia 8, ma in un recente studio, l&#39;abbattimento è stato raggiunto in A. aegypti larve immerso in una soluzione di dsRNA 15, che gli animali sembravano ingestione. Tuttavia, per gli studi che coinvolgono l&#39;analisi di molteplici experimentagruppi litri o fenotipi, immersione è piuttosto costoso. Lopez-Martinez et al. 16 descritto reidratazione guidato RNAi, un nuovo approccio per la consegna interferenza RNA che coinvolge la disidratazione in soluzione salina e reidratazione con una sola goccia di acqua contenente RNA interferenti. Questo approccio non tagliare i costi associati con tutto immersione animale, ma è più costoso di microiniezione e può essere limitata nella sua applicazione alle specie che possono tollerare alte pressioni osmotiche. Inoltre, è difficile immaginare come immersione o disidratazione / reidratazione metodologia immersione potrebbero essere adattati per il controllo vettoriale nel campo. Per queste ragioni, per studi post-embrionali, la consegna di RNA interferenti con il cibo ingerito è una strategia valida alternativa. Benché le strategie basate ingestione-non funzionano in tutte le specie di insetti, forse in particolare Drosophila melanogaster, la consegna orale di RNA interferenti mescolato al cibo ha promosso gene silencing in una varietà di insetti 8,17, comprese A. adulti aegypti 18. Abbiamo descritto chitosano nanoparticelle mediata RNAi in A. gambiae larve 19 e hanno applicato con successo questo approccio per la riduzione dell&#39;espressione genica in A. aegypti larve 20,21. Qui, metodologia per questa procedura RNAi, che comporta intrappolamento di interferire RNA dal chitosano polimero, è dettagliato. Chitosano / RNA interferenti nanoparticelle sono formate da auto-assemblaggio di policationi con interferenza RNA attraverso le forze elettrostatiche tra cariche positive dei gruppi amminici in chitosano e cariche negative portate dai gruppi fosfato sul backbone di interferire RNA 19. La procedura descritta è compatibile con entrambe le molecole dsRNA lunghi (di seguito denominati dsRNA) o doppio filamento siRNA (di seguito denominato siRNA). A seguito di sintesi, chitosano / interferenti nanoparticelle RNA sono mescolati con il cibo larvale e consegnatilarve per ingestione. Questa metodologia è relativamente poco costoso, richiede poca attrezzatura e del lavoro 19, e facilita l&#39;analisi high-throughput di più fenotipi, compresa l&#39;analisi dei comportamenti 20,21. Questa metodologia, che può essere adattato per studi silenziamento genico in altri insetti, compresi altri vettori di malattie e parassiti agricoli insetti, potrebbe essere utilizzato per il silenziamento genico in una varietà di altre specie animali. Inoltre, chitosano, un poco costoso, non tossico e biodegradabile polimero 22, potrebbe potenzialmente essere utilizzato nel campo del controllo della zanzara specie-specifico.

<table border="0" cellpadding="0" cellspacing="0" width="1429">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Name of Reagent/Equipment</td>
			<td style="width:243px;">Company</td>
			<td style="width:177px;">Catalog Number</td>
			<td style="width:663px;">Comments/Descriptions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.02 ml pipetteman</td>
			<td>Rainin</td>
			<td>PR20</td>
			<td>for resuspension of interfering RNA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.2 ml pipetteman</td>
			<td>Rainin</td>
			<td>PR200</td>
			<td>for resuspension of interfering RNA and preparation of interfering RNA/nanoparticles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.5 ml graduated tube&#160;</td>
			<td>Fischer Scientific</td>
			<td>05-408-120</td>
			<td>for aliquoting resuspended interfering RNA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 ml pipetteman</td>
			<td>Rainin</td>
			<td>PR1000</td>
			<td>for resuspension of interfering RNA and preparation of interfering RNA/nanoparticles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1.5 ml graduated tube&#160;</td>
			<td>Fischer Scientific</td>
			<td>05-408-046</td>
			<td>for preparation of interfering RNA/nanoparticles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1M Sodium Acetate, pH 4.5</td>
			<td>TEKnova</td>
			<td>S0299</td>
			<td>to prepare sodium acetate buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetic Acid, AIRSTAR. ACS, USP, FCC Grade</td>
			<td>BDH</td>
			<td>VWR BDH3092-500MLP</td>
			<td>to prepare sodium acetate buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agarose Genetic Technology Grade</td>
			<td>MP Biomedicals LLC.</td>
			<td>800668</td>
			<td>to coat prepared interfering RNA/nanoparticles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge</td>
			<td>Eppendorf AG</td>
			<td>5415D</td>
			<td>to pellet interfering RNA/nanoparticles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chitosan, from shrimp shells</td>
			<td>Sigma-Aldrich</td>
			<td>C3646-25G</td>
			<td>to combine with interfering RNA for prepararation of interfering RNA/nanoparticles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dry Yeast</td>
			<td>Universal Food Corp</td>
			<td>NA</td>
			<td>to prepare mosquito larval food with interfering RNA/nanoparticles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">E-RNAi tool</td>
			<td>German Cancer Research Center</td>
			<td>NA</td>
			<td>for design of dsRNA; http://www.dkfz.de/signaling/e-rnai3//</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">goldfish food</td>
			<td>Wardley</td>
			<td>Goldfish FLAKE FOOD</td>
			<td>to prepare mosquito larval food with interfering RNA/nanoparticles</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Heated water bath</td>
			<td>Thermo Scientific</td>
			<td>51221048</td>
			<td>to heat the interfering RNA/nanoparticles at 55oC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ice</td>
			<td>not applicable</td>
			<td>not applicable</td>
			<td>for thawing interfering RNA and preparation of interfering RNA/nanoparticles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ice bucket</td>
			<td>Fisher Scientific</td>
			<td>02-591-44</td>
			<td>for storage of ice used during the procedure</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">liver powder</td>
			<td>MP Biomedicals LLC.</td>
			<td>900396</td>
			<td>to feed mosquito larvae post interfering RNA/nanoparticle treatment</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microwave oven</td>
			<td>A variety of vendors</td>
			<td>not applicable</td>
			<td>to prepare 2% agarose solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">petridish (100X15 mm)</td>
			<td>Fischer Scientific</td>
			<td>875713</td>
			<td>interfering RNA/nanoparticle feeding chamber for larvae</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pH meter</td>
			<td>Mettler Toledo</td>
			<td>S220</td>
			<td>for preparation of buffers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Razor blade</td>
			<td>Fischer Scientific</td>
			<td>12-640</td>
			<td>to divide the interfering RNA/nanoparticle pellet for feedings</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">siRNA</td>
			<td>Thermo Scientific/Dharmacon</td>
			<td>custom</td>
			<td>for preparation of siRNA/nanoparticles</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Sodium Sulfate, Anhydrous (Na2SO4)</td>
			<td>BDH/distributed by VWR</td>
			<td>VWR BDH0302-500G</td>
			<td>to prepare 50mM sodium sulfate solution in which the interfering RNA will be resuspended</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thermometer</td>
			<td>VWR</td>
			<td>61066-046</td>
			<td>to measure the water bath temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tooth picks</td>
			<td>VWR</td>
			<td>470146-908</td>
			<td>for stirring during interfering RNA/nanoparticle food preparation and cutting gel pellets</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Ultralow freezer</td>
			<td>A variety of vendors</td>
			<td>not applicable</td>
			<td>for storage of interfering RNA aliquots and interfering RNA/nanoparticles at -80oC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vortex mixer</td>
			<td>Fischer Scientific</td>
			<td>02-216-108</td>
			<td>for preparation of chitosan/interfering RNA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Weight paper</td>
			<td>Fischer Scientific</td>
			<td>NC9798735</td>
			<td>to divide the interfering RNA/nanoparticle pellet for feedings</td>
		</tr>
	</tbody>
</table>

chitosan/interfering rna nanoparticle mediated gene silencing in disease vector mosquito larvae

Zanzare vettore infliggono sofferenze più umano di qualsiasi altro organismo - e uccidono più di un milione di persone ogni anno. I progetti genoma zanzara facilitato la ricerca in nuove sfaccettature della biologia della zanzara, inclusi studi genetici funzionali nel primario malaria vettore africano Anopheles gambiae e la dengue e la febbre gialla vettore Aedes aegypti. RNA interferenze (RNAi-) mediato silenziamento genico è stato utilizzato per colpire geni di interesse in entrambe le specie di zanzare vettori della malattia. Qui, descriviamo un procedimento per la preparazione di chitosano / interferenti nanoparticelle RNA che si combinano con il cibo e ingerite dalle larve. Questo tecnicamente semplice, ad alta velocità, e la metodologia relativamente poco costoso, che è compatibile con lunghi RNA a doppio filamento (dsRNA) o piccole interferenti RNA (siRNA) molecole, è stato utilizzato per il colpo di successo di diversi geni in A. gambiae e A. aegyptilarve. Dopo poppate larvali, atterramento, che viene verificata attraverso qRT-PCR o ibridazione in situ, possono persistere almeno fino alla fase avanzata di pupa. Questo metodo può essere applicabile ad un&#39;ampia varietà di zanzare ed altri specie di insetti infestanti, compresi agricoli, nonché altri organismi non-modello. Oltre alla sua utilità nel laboratorio di ricerca, in futuro, chitosano, un polimero poco costoso, non tossico e biodegradabile, potrebbero essere utilizzati nel campo.

<html> A. gambiae: Nanoparticelle Chitosan / dsRNA si formano a causa della interazione elettrostatica tra i gruppi amminici del chitosano ei gruppi fosfato di dsRNA. L&#39;efficienza di dsRNA incorporazione in nanoparticelle è generalmente superiore al 90% come misurato dalla deplezione del dsRNA dalla soluzione. Immagini microscopia a forza atomica mostrano che il chitosano-dsRNA medie dimensioni delle particelle 140 nm di diametro, che vanno 100-200 nm (F...

Watch this Scientific Journal Video about Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae at JoVE.com

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

Qui si descrive un procedimento per inibire la funzione del gene in zanzare vettori di malattie attraverso l&#39;uso di chitosano / RNA interferenti nanoparticelle che vengono ingeriti da larve.

Il chitosano / interferenti RNA nanoparticelle mediata silenziamento genico in Disease Vector Mosquito Larve

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects ...

chitosaninterfering-rna-nanoparticle-mediated-gene-silencing-disease

Research

JoVE Journal

Biology

14.2K Views.  Kansas State University.  Qui si descrive un procedimento per inibire la funzione del gene in zanzare vettori di malattie attraverso l'uso di chitosano / RNA interferenti nanoparticelle che vengono ingeriti da larve. 

Video: Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

Virale silenziamento genico (VIGS) in Nicotiana benthamiana E Pomodoro

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

Ricerca

Biologia

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

Vivere Imaging calcio cella Combinato con il silenziamento del gene mediata siRNA Identifica Ca 2 + Canali di perdite nella membrana ER e nei relativi meccanismi regolatori

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

Mediata da Agrobacterium Virus-Induced tacere Assay Gene In Cotone

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Isolamento dei ribosomi Polipeptidi Bound nascenti<em&gt; In vitro</em&gt; Per identificare i siti di pausa traslazione lungo mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Utilizzando Sequence SECM arresto come uno strumento per isolare ribosoma Polipeptidi Bound

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Depletion of Ribosomal 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 and functionality of the gut community will provide insight into the effects of gut microbiota on mosquito life traits. Metagenomic RNA-Seq has become an important tool to analyze transcriptomes from various microbes present in a microbial community. Messenger RNA usually comprises only 1-3% of total RNA, while rRNA constitutes approximately 90%. It is challenging to enrich messenger RNA from a metagenomic microbial RNA sample because most prokaryotic mRNA species lack stable poly(A) tails. This prevents oligo d(T) mediated mRNA isolation. Here, we describe a protocol that employs sample derived rRNA capture probes to remove rRNA from a metagenomic total RNA sample. To begin, both mosquito and microbial small and large subunit rRNA fragments are amplified from a metagenomic community DNA sample. Then, the community specific biotinylated antisense ribosomal RNA probes are synthesized in vitro using T7 RNA polymerase. The biotinylated rRNA probes are hybridized to the total RNA. The hybrids are captured by streptavidin-coated beads and removed from the total RNA. This subtraction-based protocol efficiently removes both mosquito and microbial rRNA from the total RNA sample. The mRNA enriched sample is further processed for RNA amplification and RNA-Seq.

A ribosomal RNA (rRNA) depletion protocol was developed to enrich messenger RNA (mRNA) for RNA-seq of the mosquito gut metatranscriptome. Sample specific rRNA probes, which were used to remove rRNA via subtraction, were created from the mosquito and its gut microbes. Performance of the protocol can result in the removal of approximately 90-99% of rRNA.

L&#39;esaurimento delle RNA ribosomiale per metagenomiche Gut Mosquito RNA-seq

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

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling1,2, while LRRK2 is implicated in the pathogenesis of Parkinson's disease3,4. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with 32P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing 32P-orthophosphate. The 32P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (32P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation.

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are multidomain proteins which encode both GTPase and kinase domains and which are phosphorylated in cells. Here, we present a protocol to label LRRK1 and LRRK2 in cells with 32P orthophosphate, thereby providing a means to measure their overall cellular phophorylation levels.

Metabolica Etichettatura di leucina Rich Repeat chinasi 1 e 2 con Radioactive fosfato

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Analisi globale di RNA Sintesi a livello di singola cellula seguente ipossia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down&#8217;s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.

Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.

Meiotic mandrino valutazione in mouse Ovociti mettendo a tacere siRNA-mediata

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon ...

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and diseases, such as obesity and fatty liver. Usually, lipids are stored in lipid droplets, which are the multifunctional lipid storage organelles in cells. Lipid droplets vary in size and number in different tissues and under different conditions. It has been reported that lipid droplets are tightly controlled through regulation of its biogenesis and degradation. In Drosophila melanogaster, the oenocyte is an important tissue for lipid metabolism and has been recently identified as a human liver analogue regarding lipid mobilization in response to stress. However, the mechanisms underlying the regulation of lipid droplet metabolism in oenocytes remain elusive. To solve this problem, it is of utmost importance to develop a reliable and sensitive method to directly visualize lipid droplet dynamic changes in oenocytes during development and under stressful conditions. Taking advantage of the lipophilic BODIPY 493/503, a lipid droplet-specific fluorescent dye, described here is a detailed protocol for the dissection and subsequent lipid droplet staining in the oenocytes of Drosophila larvae in response to starvation. This allows for qualitative analysis of lipid droplet dynamics under various conditions by confocal microscopy. Furthermore, this rapid and highly reproducible method can also be used in genetic screens for indentifying novel genetic factors involving lipid droplet metabolism in oenocytes and other tissues.

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Presented here are detailed methods for the dissection and lipid droplet staining of oenocytes in Drosophila larvae using BODIPY 493/503, a lipid droplet-specific fluorescent dye.

Colorazione delle goccioline per nidi e lipidi degli ovociti nelle larve di Drosophila

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and ...