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. Sivrisinek Türlerinin ve Yetiştirme <ol><li> A. koruyun gambiae G3 ve A. aegypti Liverpool IB12 (aşağıda temsili çalışmalarda kullanılan) suşları veya standart laboratuar uygulamaları ya da daha önce 23,24 açıklandığı gibi uygun diğer ilgi suşları. </li></ol> 2. dsRNA&#39;nın ve siRNA Tasarım ve Üretimi <ol><li> Tasarım primerler ilgi konusu gen için özel uzun dsRNA şablonlar oluşturmak için yerleştirildi. E-RNAi aracını 25 kullanın. Gibi bir seçim ya da yöntemine göre dsRNA üretmek önce 19 tanımladı. <ol><li> Negatif bir kontrol olarak, yeşil flüoresan proteini (GFP), β-galaktosidaz (β-gal) veya sivrisinek ifade edilmeyen başka bir gen sekansına karşılık gelen dsRNA 19 sentezler. Bu şekilde istenmesi halinde, aşağıda temsil sonuçları üretmek için kullanılan dsRNA 19, bir pozitif kontrol olarak kullanılabilir. Mağaza dsRNA&#39;nın -80 ° &#39;de RNAse içermeyen su içinde çözüldüC kadar gerekli. </li></ol></li><li> Seçenek olarak ise, tasarım gene özel siRNA standart laboratuar uygulamaya göre ya da daha önce tarif edilen, 10. Sivrisinek genine karşılık negatif kontrol siRNA tasarımı için portatif siRNA dizisini karıştırmak. Saygın satıcıları bir numara ile mevcut özel siRNA&#39;ları, satın alın. <ol><li> Bu şekilde istenmesi halinde, aşağıda temsil sonuçları elde etmek için kullanılan siRNA&#39;lar 20,21 pozitif kontrol olarak da kullanılabilir. Gerekli olana kadar mağazası siRNA -80 ° C RNAse içermeyen su içinde çözülmüştür. </li></ol></li></ol> 3. RNA Nanopartiküller müdahale / Chitosan Hazırlama <ol><li> Önceden yapılan RT 100 mM sodyum sülfat toplamak (deiyonize H 2 O 100 mM Na 2SO 4) ve 0.1 M sodyum asetat (deiyonize H 2 O 0.1 M, NAC 2H 3 O 2 -0.1 M asetik asit, pH 4.5) tamponlar. </li><li> Kitosan çözülür (≥75%deasetillenmiş), 0.1 M sodyum asetat tampon maddesi içinde () ağırlık / hacim çalışma çözeltisi% 0.02 yapmak ve kullanılmadan önce oda sıcaklığında çözelti tutun. </li><li> H2O deiyonize 50 ul dsRNA veya siRNA çözülür ve 50 mM sodyum sülfat, 100 ul başına dsRNA / siRNA 32 ug 100 ul solüsyon yapmak için, 100 mM sodyum sülfat tamponu ekleyin. </li><li> DsRNA&#39;nın / siRNA çözeltisine çitosan solüsyonu, 100 ul ilave edin ve daha sonra 1 dakika boyunca 55 ° C&#39;de bir su banyosunda karışım ısı. Kitosan çözeltisinin 100 ul 50 mM sodyum sülfat 100 ul ekleyerek bir denetim kurmak ve aynı prosedürü izleyin. </li><li> Nanopartiküllerin oluşumunu kolaylaştırmak için oda sıcaklığında yüksek hızlı bir girdaplama ile 30 saniye için hemen çözüm karıştırın. </li><li> Pelet görünür olmalıdır, bu süreden sonra, oda sıcaklığında 10 dakika boyunca 13,000 x g&#39;de santrifüjleyin karışımı. Yeni 1.5 ml tüp süpernatant aktarın. Sivrisinek yemek hazırlamak için kullanmadan önce oda sıcaklığında ≈10 dakika pelet Hava kurulayın. </li><li> Msüpernatant kaldı DsRNA / siRNA&#39;nın toplam miktarını hesaplamak için boş olarak kontrol (3.5 bakınız) süpernatant kullanarak süpernatanda DsRNA / siRNA&#39;nın konsantrasyonunu gözetim kuruluşunun onayını almıştır. Başlangıç ​​dsRNA&#39;nın / siRNA&#39;nın miktarlarda ve nanopartiküller içinde sıkışıp kalan DsRNA / siRNA&#39;nın yüzdesini hesaplamak için Süpernatantları kalan tutarları arasındaki farkı kullanın. Bu yükleme verimliliği normalde% 90 üzerindedir. </li><li> Daha fazla nanopartiküller gerekirse aynı işlemi tekrarlayın. Hemen kurutuldu nano-tanecikleri kullanın. kullanımdan önce parçacıkların soğuk depolama etkisi değerlendirilmemiştir. </li></ol> 4. Sivrisinek Gıda RNA Nanopartiküller müdahale / Chitosan içeren Hazırlanması <ol><li> Iyonu giderilmiş su içinde, agaroz eriyik (ağırlık / hacim),% 2 agaroz çözeltisi 1 ml hazırlayın ve kullanımdan önce bir 55 ° su banyosu içinde eritilmiş agaroz çözüm tutmak. </li><li> Balık gevreği karıştırın (% 47 ham protein, dk. Ham yağ% 10, maks. Ham lif% 3) en ve kuru maya(ağırlık / ağırlık) 1: 2 oranı. Bir havan ve tokmak (No 50 ABD standart test elek boyunca yeterli) küçük parçacıklara mikserden. Ya hemen, kahverengimsi renkte olmalıdır zemin yiyecek, kullanın ya da birkaç hafta boyunca 4 ˚C de kapalı bir kapta saklayın. </li><li> 1.5 ml bir tüp içinde, bir kürdan ile Bölüm 3.7 kurutulmuş nanopartiküller ile yer gıda 6 mg karıştırın. </li><li> Gıda nanopartikül karışımı,% 2, önceden eritilmiş agaroz jel çözeltisi 30 ul ilave et; Bir kürdan veya pipet ucu ile hemen ve iyice karıştırın. </li><li> Hemen sivrisinek larvaları beslemek için yiyecek ve nanopartiküller içeren jel kullanın. Jel tamamen oda sıcaklığında katılaşan bir kez, alternatif olarak, 4 ° C&#39;de jel depolama ve kullanma ertesi gün ya da daha sonra kullanmak için -80 ° C &#39;de. </li></ol> 5. Gıda RNA Nanopartiküller müdahale / Chitosan İçeren ile Sivrisinek Larva Besleme <ol><li> Besleme A. gambiae sivrisinek larvaları: <ol><li> Bir si kaldırBir kürdan kullanarak ve temiz bir jilet veya diş çekme kullanarak 6 eşit dilimler halinde kesilmiş 1.5 ml tüp ngle jel pelet. </li><li> Iyonu giderilmiş su, 100 ml içeren 500 ml&#39;lik bir Petri tabağına aktarın 20 üçüncü instar larvaları. </li><li> Petri kabı bir kez, dört gün boyunca, günde jel pellet (ince küçük parçalar halinde kesilmiş) bir dilim ekleyerek sivrisinek larvaları besleyin. Önemli ölçüde küçültülür veya ertesi gün tamamen yok edilmelidir pelet, üzerinde larva besleme gözlemlemek için emin olun. Süre sonra, sivrisinekler geç dördüncü evre larvaları dönüşecek. </li><li> Deney sırasında herhangi bir görünür fenotipik değişiklikleri kaydedin. Dört günlük sürenin sonunda 6. bölümde tartışıldığı gibi transkript düzeyleri ve diğer fenotipik değişiklikleri incelemek. </li></ol></li><li> Besleme A. aegypti sivrisinek larvaları: <ol><li> Temiz bir jilet veya kürdan kullanarak 6 eşit dilimler halinde jel pelet kesin. </li><li> Yumurta kuluçka fi sonra 50 yaş senkronize 24 saat yerleştirindeiyonize su ≈40 ml bir petri çanağı içine ilk instar larvaları. </li><li> Günün geri kalanı için 1 zemin balık yiyecek gevreği ve kuru maya: sonra 2 normal larva diyetine geri larvaları transfer 4 saat / gün petri başına larva bir dilim besleyin. Günde dört larva dönemi boyunca prosedürü tekrarlayın. </li><li> Istenen gelişimsel zaman noktalarında bölüm 6&#39;da anlatıldığı gibi transkript düzeyleri ve diğer fenotipik değişiklikleri incelemek. </li></ol></li></ol> Gen demonte 6. Onay <ol><li> A. Mah-PCR ile Bağıl transkript ölçümü aegypti ve A. gambiae larvaları. <ol><li> Gerçekleştirin ve 11,19,20 tarif, ya da standart laboratuar prosedüre göre deney larva vs en az 10 havuza kontrol üç biyolojik çoğaltır ile Mah-PCR deneyleri analiz. Örnek sonuçlar aşağıda yer almaktadır. </li><li> Özel bir doku tipi / gövde parçası (örn. Beyin veya anten), perfo analizi içinÇevrede bulunan doku kurtarma 6.1.1 aşağıdaki diseksiyon tarif edildiği gibi rm QRT-PCR (örneğin, bakınız Mysore ve ark. 21). </li></ol></li><li> In situ olarak melezleşme ile demonte teyidi <ol><li> Dioksijenin-etiketli antisens sentez ve standart laboratuar uygulamaları ya da 26 anlatıldığı gibi uygun kontrol riboprobes seziyorum. </li><li> Daha önce 11,20 açıklandığı ve aşağıda temsilcisi sonuç bölümünde tartışıldığı gibi demonte teyidi için Haugen ark. 27 protokol başına in situ hibridizasyon yürütün. </li></ol></li><li> Immünohistokimyasal olarak demonte Onayı <ol><li> Hedeflenmiş genin protein ürünü karşı antikorların mevcut ise, daha önce tarif edildiği gibi 28, representati örneklendiği gibi fenotip analizi 20 demonte büyük seviyeleri olan bireylerin belirlenmesi kolaylaştıran, immünohistokimya gerçekleştirmekAşağıdaki sonuçlar bölümünde ettik. </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.

Burada tarif edilen çitosan / müdahale edici RNA nanopartikül yöntem etkili bir şekilde A. larva gelişimi sırasında hedef genlere kullanılmıştır gambiae (Şekil 2, 3) ve A. aegypti (Şekiller 4, 5, 6, Tablolar 1, 2). Kitosan nanopartiküller olarak burada tarif edilen sonuçları ile kanıtlandığı gibi, sivrisinek olarak başarılı bir şekilde kullanılmıştır, her ikisi de uzun dsRNA veya siRNA, ya da hazırlanabilir. DsRNA&#39;nın Sentezi...

Anopheline türü ve aedine cins Kan besleme vektör sivrisinek insanlığın en kötü bela birkaç sorumlu hastalığa neden olan ajanlar iletir. Tahminen 3,4 milyar insan yılda dünya çapında üzerinden bir buçuk milyon kişinin ölümünden sorumlu olan sıtma, sözleşme için risk altındadır. Plasmodium sp enfeksiyonundan Sıtma sonuçları. Başlıca Afrika vektörü Anopheles gambiae (dahil Anopheles cinsi sivrisineklerin enfekte, ısırıkları ile insanlara iletilir parazitler, <a href="http://www.who.int/topics/malaria/en/">http://www.who.int/topics/malaria/en/</a> , 2014) 1. Aedes aegypti dang ateşi, dünyanın en yaygın ve önemli arboviral hastalıktır nonspesifik ateşli hastalığa neden dang virüsü, birincil sivrisinek vektör. Dang virüsü yıllık inciden ile, halen tropik&gt; 2,5 milyar kişiye bir tehdittir24.000 ölüm yıl ~ sonuçlanan yaklaşık 50 milyon olgunun ce ( <a href="http://www.cdc.gov/dengue/">http://www.cdc.gov/dengue/</a> , 2014) 2. Insan sağlığı üzerindeki sivrisinek yoluyla bulaşan hastalıkların yıkıcı küresel etkilerine rağmen, önlenmesi ve bu hastalıkların tedavisinde etkili aracı eksiktir. Sivrisinek kontrolü halen hastalığın önlenmesi en iyi yöntemdir. Vektör böceklerin genetik manipülasyon yoluyla Artropod kaynaklı hastalıkların kontrolü için potansiyeli dört yılı aşkın bir süredir 3 için kabul edilmiştir. A. Transgenik soylar Son zamanlarda transgenik vektör kontrol stratejileri gerçeğe 4-6 kullanarak potansiyelini yaptık aegypti bir bastırılabilir kadın-özel uçamayan fenotip sahip tasarlanmış. Bu gelişmeler vektör kontrolü ve vektör sivrisinek gen fonksiyonunu manipüle ek araçları için yeni genetik hedefler belirlemek için araştırmacılar meydan var. Gen ifadesi d Tadilatınakadın-uçamayan sivrisinekler 4 yılında olduğu gibi uring geliştirme, yeni vektör kontrol stratejilerinin açıklanmasını teşvik edebilir. Ancak, büyük ölçüde teknik zorluklar, çok az genlerin fonksiyonları A. geliştirilmesi sırasında karakterize edilmiştir gambiae, A. aegypti veya diğer sivrisinek türleridir. C yılında keşfedilmesinden bu yana elegans 7, hayvanlar, bitkiler ve mikroorganizmalar muhafaza edilir, RNA interferans (RNAi), yaygın olarak böcekler 8,9 dahil olmak üzere, geniş bir organizma, çeşitli işlevsel genetik çalışmalar için kullanılmıştır. RNAi yolu diziye özgü müdahale RNA olarak işlev kısa 20-25 nükleotid uzunluğundaki siRNA&#39;lar içine Dicer bölünen uzun dsRNA ile başlatılır. transkript ciro, bölünme ve çeviri 9 bozulması teşvik ederek sırayla tamamlayıcı siRNA&#39;lar sessizlik genler. Bir Particula hedefleme uzun dsRNA molekülleri (tipik olarak 300-600 bp) ya da özel siRNA&#39;larR dizisi, ilgi konusu herhangi bir gen susturma araştırma laboratuarda kullanılabilir. RNA teslim edilir müdahale sırasında yöneterek, araştırmacılar hangi gen de inisiyelere susturulması zaman kontrol edebilirsiniz. Bu üretim ve kalıtsal mutasyonlar, henüz tüm böcek türlerinin mevcut değildir pahalı ve emek-yoğun bir süreç taşıyan suşların bakım engelleyebilir gibi gelişimsel öldürücülüğü veya kısırlık gibi zorlukların üstesinden gelmek için kullanılabildiği gibi bu avantaj yararlıdır. RNAi, gen susturma derecesi hayvana geni, dokuya doku ve hayvan geninin arasında değişebilir, ancak, RNAi yaygın sivrisinek ve diğer böcekler 8,9 genlerin işlevsel analiz için kullanılmaktadır. Üç müdahale RNA teslim stratejileri sivrisinekler kullanılmıştır: mikroenjeksiyon, iliklerine / topikal uygulama, ve yenmesi. Detaylı bir tarih ve böcekler bu üç teknikler kullanılarak Karşılaştırma için, Yu ve arkadaşları 8 bakınız. WE başarılı bir şekilde A. gelişim hedef genlere siRNA&#39;lar vermek için bir araç olarak mikroenjeksiyon 10 kullandık aegypti embriyolar, larva, pupa ve 11-14. Ancak, bu emek-yoğun teslimat strateji mikroenjeksiyon kurulum ve yetenekli bir el hem de gerektirir. Ayrıca, mikroenjeksiyon davranışsal fenotipleri değerlendirilecek, özellikle organizma, bir karıştırıcı faktör streslidir. Son olarak, mikro-enjeksiyon aktarım vektörü kontrol alanına uzatılamaz. Bu uygundur ve küçük ekipman veya emek gerektirir bir alternatif olarak, RNA çözümü müdahale organizmayı iliklerine de, gen susturulması tetikleme popüler bir araç haline gelmiştir. Islatma öncelikle böcek hücre hattında uygulanmaktadır 8 çalışmaları, ancak son çalışmada, Knockdown A&#39;da elde edildi aegypti larva hayvanlar sindirerek ortaya çıktı dsRNA 15, ihtiva eden bir çözeltiye batırılır. Bununla birlikte, birden fazla experimenta analizini kapsayan çalışmalar içinL grubu veya fenotipleri, ıslatma oldukça maliyetlidir. Lopez-Martinez ve diğ. 16 RNAi, engelleyen RNA ihtiva eden tek bir su damlası ile tuzlu su çözeltisi ve rehidrasyon dehidratasyon içeren RNA teslim müdahale için yeni bir yaklaşım tahrik rehidrasyon tarif. Bu yaklaşım, tüm hayvan daldırma ile ilişkili maliyetlerini düşürmek, ancak mikroenjeksiyon daha pahalı ve yüksek ozmotik basınçları tolere edebilir türlere uygulanması sınırlı edilebilir değildir. Ayrıca, daldırma ya da dehidrasyon / rehidrasyon daldırma yöntemi alanında vektör kontrolü için uyarlanabilir nasıl tasavvur etmek zordur. Bu nedenlerden dolayı, post-embriyonik çalışmalar için alınan gıda ile müdahaleci RNA teslim uygun bir alternatif bir stratejidir. Yenmesi-tabanlı stratejiler, tüm böcek türleri, belki de en önemlisi Drosophila melanogaster çalışmıyor rağmen, gıda ile karışık RNA müdahale oral teslim gen si terfi ettiBöceklerin 8,17 çeşitli lencing, aşağıdakileri içeren A. aegypti yetişkin 18. Bu A&#39;da kitosan nanopartikül aracılı RNAi tarif gambiae larva 19 ve başarılı bir şekilde A. gen ekspresyonunun azaltılması için, bu yaklaşım uyguladık aegypti larvaları 20,21. Burada, polimer kitosan ile RNA müdahale sürüklenmemesine içerir bu RNAi prosedürü, metodolojisi, ayrıntılı. Kitosan / müdahale edici RNA nanopartiküller çitosan amino gruplarının pozitif yüklerin ve RNA 19 müdahale omurgasında fosfat grupları tarafından taşınan negatif yük arasındaki elektrostatik kuvvetler aracılığıyla müdahaleci RNA ile polikatyonlar kendini düzenlemesinden oluşur. açıklanan prosedür uzun dsRNA moleküllerinin (bundan sonra siRNA olarak anılacaktır) siRNA şeritli (bundan sonra dsRNA olarak anılacaktır) ya da çift ile uyumludur. Aşağıdaki sentez, çitosan / müdahale edici RNA nanopartiküller Konukçu ile karıştırıldı ve teslim edilirOral yoldan alım yoluyla larva. Bu metodoloji, nispeten ucuz küçük ekipman ve emek gerektirir 19, ve davranışların 20,21 analizi de dahil olmak üzere çok sayıda fenotip, yüksek verimlilik analizini kolaylaştırır. Diğer hastalık vektörleri ve böcek tarım haşere de dahil olmak üzere diğer böcekler, gen susturma çalışmaları için adapte edilebilir Bu metodoloji, potansiyel olarak diğer hayvan türlerinin çeşitli gen susturma için kullanılabilir. Ayrıca, çitosan, ucuz, toksik olmayan ve biyolojik olarak parçalanabilir bir polimer 22, potansiyel olarak türe özgü sivrisinek kontrolü için alanda kullanılabilir.

<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

Vektör Sivrisinek başka bir organizma daha çok insan acı indirebilmek - ve bir milyondan fazla insanı her yıl öldürmek. Sivrisinek genom projeleri birincil Afrika sıtma vektörü Anopheles gambiae ve dang ve sarı humma vektör Aedes aegypti fonksiyonel genetik çalışmalar da dahil olmak üzere sivrisinek biyoloji yeni yönleriyle, araştırma kolaylaştırdı. RNA parazitsiz (RNAi-) aracılık ettiği gen susması, bu hastalık taşıyıcıları sivrisinek türlerinin her ikisi de ilgi genleri hedef olarak kullanılmaktadır. Burada, gıda ile birlikte ve larvaları tarafından sindirilen RNA nano-tanecikleri müdahale / çitosan hazırlanması için bir prosedür tarif eder. Uzun çift kollu RNA (dsRNA), ya da küçük müdahale edici RNA (siRNA) molekülleri ile uyumlu olan bu teknik basit, yüksek hacimli, ve nispeten pahalı olmayan bir yöntem, A. farklı geninin başarılı bir şekilde demonte kullanılmaktadır gambiae ve A. aegyptiMah-PCR ile veya in situ hibridizasyon doğrulandı larva. larva beslenmeye ardından, demonte, en azından geç pupa aşamasından devam edebilirsiniz. Bu yöntem, bir sivrisinek ve tarımsal zararlıların da dahil olmak üzere diğer böcek türleri, çok çeşitli, hem de diğer olmayan modeli organizmalar için de geçerli olabilir. Araştırma laboratuarında de yararlı ek olarak, gelecekte, çitosan, ucuz, toksik olmayan ve biyolojik olarak parçalanabilir bir polimer içinde, potansiyel alanında kullanılabilir.

<html> A. gambiae: Kitosan / dsRNA&#39;nın nanopartiküller bağlı çitosan amino grupları ve dsRNA&#39;nın fosfat grupları arasındaki elektrostatik etkileşim oluşur. çözeltiden dsRNA&#39;nın azalması ile ölçülen nanopartiküller olarak dsRNA dahil verimliliği genellikle% 90 üzerindedir. Atomik kuvvet mikroskopisi görüntüler şunu gösterir ki, 100-200 nm&#39;de (Şekil 1) arasında değişen çap olarak kitosan-dsRNA parçacık b...

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

Burada larvaları tarafından sindirilen RNA nano-tanecikleri / kitosan kullanımı yoluyla hastalık taşıyıcıları sivrisinek gen fonksiyonunun inhibe edilmesine müdahale için bir prosedür tarif eder.

Hastalık Vektör Sivrisinek Larva içinde Kitosan / Müdahale RNA Nanopartikül Aracılı gen susturma

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

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14.2K Görüntüleme.  Kansas State University.  Burada larvaları tarafından sindirilen RNA nano-tanecikleri / kitosan kullanımı yoluyla hastalık taşıyıcıları sivrisinek gen fonksiyonunun inhibe edilmesine müdahale için bir prosedür tarif eder. 

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.

Virüs indüklenen gen susturma (VIGS) Nicotiana benthamiana Ve Domates

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

Biyoloji

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.

Canlı Hücre Kalsiyum Görüntüleme siRNA aracılığı ile gen susturma ile birlikte Ca tanımlar 2 + ER Membran Kaçak Kanallar ve Düzenleme Mekanizmaları

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.

Pamuk olarak Agrobacterium Virüs Bağlı Gen susturma Testi

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.

Ribozom Bağlı Doğan Polipeptitler İzolasyonu<em&gt; In vitro</em&gt; MRNA boyunca Translasyonel Duraklat Siteleri Belirleme

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.

Ribozom Ciltli Polipeptitler izole bir aracı olarak SECM Tutuklanma sırası kullanılarak

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.

Sivrisinek Gut Metagenomic RNA-seq Ribozomal RNA tükenmesi

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.

Lösin zengin metabolik etiketleme Fosfat ile radyoaktif Kinazlar 1 ve 2, tekrar

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.

Ününüz Aşağıdaki Tek Hücre Düzeyinde Küresel RNA Sentezi Analizi

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.

SiRNA aracılı susturma Mouse Oositlerinin içinde mayoz Mili Değerlendirmesi

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.

Drosophila Larvalarında Oenositlerin Diseksiyonu ve Lipid Damlacık Boyaması

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