Name: Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae (Video) | JoVE Journal
Uploaded: 2015-03-25T13:00:00.000+00:00
Duration: 8 min 36 s
Description: Learn chitosan/interfering RNA nanoparticle gene silencing in disease vector mosquito larvae. This method enables functional studies of genes in Anopheles gambiae and Aedes aegypti using oral delivery.

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5 minutes

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 Species and Rearing
<ol>
	<li>Maintain A. gambiae G3 and A. aegypti&#160;Liverpool IB12 strains (used in the representative studies below) or other strains of interest according to standard lab practice or as described previously23,24.</li>
</ol>
2. dsRNA and siRNA Design and Production
<ol>
	<li>Design primers to construct long dsRNA templates specific to the gene of interest. Use the E-RNAi tool25. Produce dsRNA according to the method of choice or as previously described19.
	<ol>
		<li>For a negative control, synthesize dsRNA19 corresponding to the sequence of green fluorescent protein (GFP), &#946;-galactosidase (&#946;-gal), or some other gene not expressed in mosquitoes. If so desired, dsRNA19 utilized to generate the representative results summarized below can be used as a positive control. Store dsRNA dissolved in RNAse-free water at -80 &#176;C until needed.</li>
	</ol>
	</li>
	<li>Alternatively, design gene-specific siRNA according to standard lab practice or as described previously10. Scramble the sequence of a knockdown siRNA to design negative control siRNA that does not correspond to any mosquito gene. Purchase the custom siRNAs, which are available through a number of reputable vendors.
	<ol>
		<li>If so desired, siRNAs20,21utilized to obtain the representative results summarized below can be used as positive controls. Store siRNA dissolved in RNAse-free water at -80 &#176;C until needed.</li>
	</ol>
	</li>
</ol>
3. Preparing Chitosan/interfering RNA Nanoparticles
<ol>
	<li>Gather pre-made RT 100 mM sodium sulfate (100 mM Na2SO4 in deionized H2O) and 0.1 M sodium acetate (0.1&#160;M NaC2H3O2&#8211;0.1&#160;M acetic acid, pH 4.5 in deionized H2O) buffers.</li>
	<li>Dissolve chitosan (&#8805;75% deacetylated) in 0.1 M sodium acetate buffer to make 0.02% (w/v) working solution and keep the solution at RT before use.</li>
	<li>Dissolve dsRNA or siRNA in 50 &#181;l deionized H2O and add it to the 100 mM sodium sulfate buffer to make a 100 &#181;l solution of 32 &#181;g of dsRNA/siRNA per 100 &#181;l of 50 mM sodium sulfate.</li>
	<li>Add 100 &#181;l of chitosan solution to the dsRNA/siRNA solution and then heat the mixture in a water bath at 55 &#176;C for 1&#160;min. Set up a control by adding 100 &#181;l of 50 mM sodium sulfate to 100 &#181;l of chitosan solution and follow the same procedure.</li>
	<li>Mix the solutions immediately for 30 sec by high-speed vortexing at RT to facilitate the formation of nanoparticles.</li>
	<li>Centrifuge the mixture at 13,000 x g for 10 min at RT, after which time a pellet should be visible. Transfer the supernatant to a new 1.5 ml tube. Air-dry the pellet for &#8776;10 min at RT before using it to prepare mosquito food.</li>
	<li>Measure the concentration of dsRNA/siRNA in the supernatant by using the supernatant from the control (see 3.5) as a blank to calculate the total amount of dsRNA/siRNA that remained in the supernatant. Use the difference between starting amounts of dsRNA/siRNA and amounts remaining in the supernatants to calculate the percentage of dsRNA/siRNA entrapped in the nanoparticles. This loading efficiency is normally over 90%.</li>
	<li>Repeat the same procedure if more nanoparticles are needed. Use the dried nanoparticles immediately. The impact of cold storage of particles prior to use has not been evaluated.</li>
</ol>
4. Preparing Mosquito Food containing Chitosan/interfering RNA Nanoparticles
<ol>
	<li>Prepare 1 ml of a 2% agarose solution (w/v) in deionized water, melt the agarose, and keep melted agarose solution in a 55 &#730;C water bath before use.</li>
	<li>Mix fish food flakes (47% crude protein, min. crude fat 10%, max. crude fiber 3%) and dry yeast at a ratio of 2:1 (w/w). Grind the mixture to small particles with a mortar and pestle (passable through No. 50 USA standard test sieve). Use the ground food, which should be brownish in color, either immediately or store it in a sealed container at 4 &#730;C for several weeks.</li>
	<li>In a 1.5 ml tube, mix 6 mg of ground food with the dried nanoparticles from Section 3.7 with a toothpick.</li>
	<li>Add 30&#160;&#181;l of 2% pre-melted agarose gel solution to the food-nanoparticle mixture; stir immediately and thoroughly by using a toothpick or pipet tip.</li>
	<li>Use the gel containing food and nanoparticles to feed the mosquito larvae immediately. Alternatively, once the gel is completely solidified at RT, store the gel at 4 &#176;C and use the next day, or at -80 &#176;C for later use.</li>
</ol>
5. Feeding Mosquito Larvae with Food Containing Chitosan/interfering RNA Nanoparticles
<ol>
	<li>Feeding A. gambiae mosquito larvae:
	<ol>
		<li>Remove a single gel pellet from the 1.5 ml tube using a toothpick and cut it into 6 equal slices using a clean razor blade or tooth pick.</li>
		<li>Transfer 20 third instar larvae to a 500 ml Petri dish containing 100 ml of deionized water.</li>
		<li>Feed the mosquito larvae by adding one slice of the gel pellet (finely chopped into smaller pieces) per Petri dish once a day for four days. Be sure to observe larvae feeding on the pellet, which should be significantly reduced in size or completely absent by the next day. After time, mosquitoes will develop into late fourth instar larvae.</li>
		<li>Record any visible phenotypic changes during the experiment. Examine the transcript levels and other phenotypic changes as discussed in section 6 at the end of the four day period.</li>
	</ol>
	</li>
	<li>Feeding A. aegypti mosquito larvae:
	<ol>
		<li>Cut the gel pellet into 6 equal slices using a clean razor blade or toothpick.</li>
		<li>Place 50 age-synchronized 24 hr after egg hatching first instar larvae into a petri dish in &#8776;40 ml deionized water.</li>
		<li>Feed larvae one slice per petri dish for 4 hr/day, then transfer larvae back to the regular larval diet of 2:1 ground fish food flakes and dry yeast for the rest of the day. Repeat procedure daily throughout the four larval instars.</li>
		<li>Examine the transcript levels and other phenotypic changes as discussed in section 6 at the desired developmental time points.</li>
	</ol>
	</li>
</ol>
6. Confirmation of Gene Knockdown
<ol>
	<li>Relative transcript quantification by qRT-PCR in A. aegypti and A. gambiae larvae.
	<ol>
		<li>Perform and analyze qRT-PCR assays with three biological replicates on at least 10 pooled control vs. experimental larvae as described11,19,20 , or according to standard lab procedure. Representative results are included below.</li>
		<li>For analysis of a particular tissue type/body part (i.e., brain or antenna), perform qRT-PCR as described in 6.1.1 following dissection to recover the tissue of interest (for example, see Mysore et al.21).</li>
	</ol>
	</li>
	<li>Confirmation of knockdown by in situ hybridization
	<ol>
		<li>Synthesize digoxygenin-labeled antisense and sense control riboprobes according to standard lab practice or as described26.&#160;</li>
		<li>Execute in situ hybridization per the Haugen et al.27 protocol for confirmation of knockdown as described previously11,20 and discussed in the representative results section below.</li>
	</ol>
	</li>
	<li>Confirmation of knockdown by immunohistochemistry
	<ol>
		<li>If antibodies against the protein product of the gene targeted are available, perform immunohistochemistry as previously described28, which facilitates identification of individuals with the greatest levels of knockdown for phenotype analysis20 as exemplified in the representative results section below.</li>
	</ol>
	</li>
</ol>

<ol><li>Malaria. , World Health Organization. 
 
 <a target="_blank" href="http://www.who.int/topics/malaria/en">http://www.who.int/topics/malaria/en</a> (2014).</li>
<li>Dengue. , Malaria Centers for Disease Control and Prevention. 
 
 <a target="_blank" href="http://www.cdc.gov/dengue/">http://www.cdc.gov/dengue/</a> (2014).</li>
<li>Knipling, E. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+control+of+insects+of+public+health+importance%5BTitle%5D)+AND+%22Bulletin+of+the+World+Health+Organization%22%5BJournal%5D)">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/pubmed?term=((Female-specific+flightless+phenotype+for+mosquito+control%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">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/pubmed?term=((Genetic+elimination+of+dengue+vector+mosquitoes%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">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/pubmed?term=((Successful+suppression+of+a+field+mosquito+population+by+sustained+release+of+engineered+male+mosquitoes%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">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/pubmed?term=((Potent+and+specific+genetic+interference+by+double-stranded+RNA+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Delivery+of+dsRNA+for+RNAi+in+insects%3A+an+overview+and+future+directions%5BTitle%5D)+AND+%22Insect+science%22%5BJournal%5D)">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/pubmed?term=((limitation+and+possible+solutions+of+RNAi-based+technology+for+insect+pest+control%5BTitle%5D)+AND+%22Insect+science%22%5BJournal%5D)">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/pubmed?term=((Functional+analysis+of+genes+in+Aedes+aegypti+embryos%5BTitle%5D)+AND+%22Cold+Spring+Harb+Protoc%22%5BJournal%5D)">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/pubmed?term=((siRNA-mediated+gene+targeting+in+Aedes+aegypti+embryos+reveals+that+frazzled+regulates+vector+mosquito+CNS+development%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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/pubmed?term=((Semaphorin-1a+is+required+for+Aedes+aegypti+embryonic+nerve+cord+development%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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/pubmed?term=((Functional+genetic+characterization+of+salivary+gland+development+in+Aedes+aegypti%5BTitle%5D)+AND+%22EvoDevo%22%5BJournal%5D)">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/pubmed?term=((Requirement+for+commissureless2+function+during+dipteran+insect+nerve+cord+development%5BTitle%5D)+AND+%22Developmental+dynamics%3A+an+official+publication+of+the+American+Association+of+Anatomists%22%5BJournal%5D)">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/pubmed?term=((Oral+delivery+of+double-stranded+RNA+in+larvae+of+the+yellow+fever+mosquito%2C+Aedes+aegypti%3A+implications+for+pest+mosquito+control%5BTitle%5D)+AND+%22J+Insect+Sci%22%5BJournal%5D)">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/pubmed?term=((Rehydration+driven+RNAi%3A+a+novel+approach+for+effectively+delivering+dsRNA+to+mosquito+larvae%5BTitle%5D)+AND+%22Journal+of+medical+entomology%22%5BJournal%5D)">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/pubmed?term=((Towards+the+elements+of+successful+insect+RNAi%5BTitle%5D)+AND+%22Journal+of+insect+physiology%22%5BJournal%5D)">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/pubmed?term=((Gene+silencing+in+adult+Aedes+aegypti+mosquitoes+through+oral+delivery+of+double-stranded+RNA%5BTitle%5D)+AND+%22J+Appl+Entomol%22%5BJournal%5D)">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/pubmed?term=((Chitosan%2Fdouble-stranded+RNA+nanoparticle-mediated+RNA+interference+to+silence+chitin+synthase+genes+through+larval+feeding+in+the+African+malaria+mosquito+%28Anopheles+gambiae%29%5BTitle%5D)+AND+%22Insect+molecular+biology%22%5BJournal%5D)">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/pubmed?term=((Disruption+of+Aedes+aegypti+olfactory+system+development+through+chitosan%2FsiRNA+nanoparticle+targeting+of+semaphorin-1a%5BTitle%5D)+AND+%22PLoS+neglected+tropical+diseases%22%5BJournal%5D)">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/pubmed?term=((Chitosan%2FsiRNA+nanoparticle+targeting+demonstrates+a+requirement+for+single-minded+during+larval+and+pupal+olfactory+system+development+of+the+vector+mosquito+Aedes+aegypti%5BTitle%5D)+AND+%22BMC+developmental+biology%22%5BJournal%5D)">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/pubmed?term=((Chitosan-mediated+orally+delivered+nucleic+acids%3A+a+gutful+of+gene+therapy%5BTitle%5D)+AND+%22Journal+of+drug+targeting%22%5BJournal%5D)">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/pubmed?term=((Characterization+of+a+regulatory+unit+that+controls+melanization+and+affects+longevity+of+mosquitoes%5BTitle%5D)+AND+%22Cellular+and+molecular+life+sciences+%3A+CMLS%22%5BJournal%5D)">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. Culturing and egg collection of Aedes aegypti. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aClemons%2C+A+%22Cold+Spring+Harb+Protoc%22">Cold Spring Harb Protoc</a>. 2010 (10), (2010).</li>
<li>Horn, T., Boutros, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((E-RNAi%3A+a+web+application+for+the+multi-species+design+of+RNAi+reagents--2010+update%5BTitle%5D)+AND+%22Nucleic+acids+research%22%5BJournal%5D)">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. Ch. 19. In situ hybridization to whole mount Drosophila embryos. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPatel%2C+NH+%22A+laboratory+guide+to+RNA%3A+Isolation%2C+Analysis%2C+and+Synthesis%22">A laboratory guide to RNA: Isolation, Analysis, and Synthesis</a>. PA, K. rieg , Wiley-Liss. (1996).</li>
<li>Haugen, M., et al. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aHaugen%2C+M+%22Whole-mount+in+situ+hybridization+for+analysis+of+gene+expression+during+Aedes+aegypti+development%22">Whole-mount in situ hybridization for analysis of gene expression during Aedes aegypti development</a>. 2010 (10), Cold Spring Harb Protoc. (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/pubmed?term=((Immunohistochemical+analysis+of+protein+expression+during+Aedes+aegypti+development%5BTitle%5D)+AND+%22Cold+Spring+Harb+Protoc%22%5BJournal%5D)">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/pubmed?term=((Genome-wide+profiling+of+diel+and+circadian+gene+expression+in+the+malaria+vector+Anopheles+gambiae%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">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. 3rd <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ZFN%2C+TALEN%2C+CRISPR%2FCas-based+methods+for+genome+engineering%5BTitle%5D)+AND+%22Trends+in+biotechnology%22%5BJournal%5D)">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/pubmed?term=((Targeted+genome+editing+in+Aedes+aegypti+using+TALENs%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">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/pubmed?term=((Oral+administration+of+bacterially+expressed+VP28dsRNA+to+protect+Penaeus+monodon+from+white+spot+syndrome+virus%5BTitle%5D)+AND+%22Mar+Biotechnol+%28NY%29%22%5BJournal%5D)">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/pubmed?term=((Fluorescent+nanoparticle+delivered+dsRNA+toward+genetic+control+of+insect+pests%29%5BTitle%5D)+AND+%22Adv+Mater%22%5BJournal%5D)">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/pubmed?term=((Gene+regulation+with+polyvalent+siRNA-nanoparticle+conjugates%5BTitle%5D)+AND+%22Journal+of+the+American+Chemical+Society%22%5BJournal%5D)">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/pubmed?term=((Evidence+of+RNAi+in+humans+from+systemically+administered+siRNA+via+targeted+nanoparticles%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Polyethylenimine+and+chitosan+carriers+for+the+delivery+of+RNA+interference+effectors%5BTitle%5D)+AND+%22Expert+opinion+on+drug+delivery%22%5BJournal%5D)">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/pubmed?term=((Polymers+in+small-interfering+RNA+delivery%5BTitle%5D)+AND+%22Nucleic+acid+therapeutics%22%5BJournal%5D)">Polymers in small-interfering RNA delivery.</a> Nucleic acid therapeutics. 21 (3), 133-148 (2011).</li>
</ol>

The authors have nothing to disclose.

The chitosan/interfering RNA nanoparticle methodology described herein has been used to effectively target genes during larval development in A. gambiae (Figures&#160;2, 3) and A. aegypti (Figures 4, 5,&#160;6, Tables 1, 2). Chitosan nanoparticles can be prepared with either long dsRNA or siRNA, both of which have been used successfully in mosquitoes as evidenced by the representative results described herein. Synthesis of dsRNA is less...

Blood feeding vector mosquitoes of the Anopheline and Aedine genera transmit disease-causing agents responsible for several of the worst scourges of humankind. An estimated 3.4 billion people are at risk for contracting malaria, which is responsible for over one-half million deaths annually worldwide. Malaria results from infection by Plasmodiumsp. parasites, which are transmitted to people through the bites of infected mosquitoes of the Anopheles genus, including the principal African vector Anopheles gambiae (<a href="http://www.who.int/topics/malaria/en/">http://www.who.int/topics/malaria/en/</a>, 2014)1. Aedes aegypti is the primary mosquito vector for dengue virus, which causes dengue fever, a nonspecific febrile illness that is the most widespread and significant arboviral disease in the world. Dengue virus is presently a threat to &#62;2.5 billion people in the tropics, with a yearly incidence of approximately 50 million cases resulting in ~24,000 deaths annually (<a href="http://www.cdc.gov/dengue/">http://www.cdc.gov/dengue/</a>, 2014)2. Despite the devastating global impact of mosquito-borne illnesses on human health, effective means of preventing and treating these diseases are lacking. Mosquito control is presently the best method of disease prevention.
The potential for controlling arthropod-borne diseases by the genetic manipulation of vector insects has been recognized for over four decades3. Transgenic strains of A. aegypti engineered to have a repressible female-specific flightless phenotype have recently made the potential for using transgenic vector control strategies a reality4-6. These advancements have challenged researchers to identify novel genetic targets for vector control and additional means of manipulating gene function in vector mosquitoes. Alteration of gene expression during development, as was the case in female-flightless mosquitoes4, may promote the elucidation of novel vector control strategies. However, largely due to technical challenges, the functions of very few genes have been characterized during development of A. gambiae, A. aegypti, or other mosquito species.
Since its discovery in C. elegans7, RNA interference (RNAi), which is conserved in animals, plants, and microorganisms, has been extensively used for functional genetic studies in a wide variety of organisms, including insects8,9. The RNAi pathway is initiated by Dicer, which cleaves long dsRNA into short 20-25 nucleotide-long siRNAs that function as sequence-specific interfering RNA. siRNAs silence genes that are complementary in sequence by promoting transcript turnover, cleavage, and disruption of translation9. Long dsRNA molecules (typically 300-600 bp) or custom siRNAs targeting a particular sequence can be used in the research laboratory for silencing any gene of interest. By managing when interfering RNA is delivered, researchers can control the time at which gene silencing initiates. This advantage is useful as it can be used to overcome challenges such as developmental lethality or sterility, which can hinder the production and maintenance of strains bearing heritable mutations, an expensive and labor-intensive process that is not yet available in all insect species. Although the degree of gene silencing by RNAi can vary from gene to gene, tissue to tissue, and animal to animal, RNAi is widely used for functional analysis of genes in mosquitoes and other insects8,9.
Three interfering RNA delivery strategies have been used in mosquitoes: microinjection, soaking/topical application, and ingestion. For a detailed history and comparison of the use of these three techniques in insects, please refer to Yu et al8. We have successfully used microinjection10 as a means of delivering siRNAs to target developmental genes in A. aegypti embryos, larvae, and pupae11-14. However, this labor-intensive delivery strategy requires both a microinjection setup and a skilled hand. Moreover, microinjection is stressful to the organism, a confounding factor, particularly when behavioral phenotypes will be assessed. Finally, microinjection delivery cannot be extended to the field for vector control. As an alternative, soaking the organism in interfering RNA solution has also become a popular means of inducing gene silencing, as it is convenient and requires little equipment or labor. Soaking has primarily been applied in insect cell line studies8, but in a recent study, knockdown was achieved in A. aegypti larvae immersed in a solution of dsRNA15, which the animals appeared to be ingesting. However, for studies involving analysis of multiple experimental groups or phenotypes, soaking is rather costly. Lopez-Martinez et al.16 described rehydration driven RNAi, a novel approach for interfering RNA delivery that involves dehydration in saline solution and rehydration with a single drop of water containing interfering RNA. This approach does cut the costs associated with whole animal immersion, but is more expensive than microinjection and may be limited in its application to species that can tolerate high osmotic pressures. Moreover, it is difficult to envision how immersion or dehydration/rehydration immersion methodology could be adapted for vector control in the field. For these reasons, for post-embryonic studies, delivery of interfering RNA with ingested food is a viable alternative strategy.
Although ingestion-based strategies do not work in all insect species, perhaps most notably Drosophila melanogaster, oral delivery of interfering RNA mixed with food has promoted gene silencing in a variety of insects8,17, including A. aegypti adults18. We described chitosan nanoparticle-mediated RNAi in A. gambiae larvae19 and have successfully applied this approach for reduction of gene expression in A. aegypti larvae20,21. Here, methodology for this RNAi procedure, which involves entrapping of interfering RNA by the polymer chitosan, is detailed. Chitosan/interfering RNA nanoparticles are formed by self-assembly of polycations with interfering RNA through the electrostatic forces between positive charges of the amino groups in chitosan and negative charges carried by the phosphate groups on the backbone of interfering RNA19. The procedure described is compatible with both long dsRNA molecules (hereafter referred to as dsRNA) or double stranded siRNA (hereafter referred to as siRNA). Following synthesis, chitosan/interfering RNA nanoparticles are mixed with larval food and delivered to larvae through oral ingestion. This methodology is relatively inexpensive, requires little equipment and labor19, and facilitates high-throughput analysis of multiple phenotypes, including analysis of behaviors20,21. This methodology, which can be adapted for gene silencing studies in other insects, including other disease vectors and insect agricultural pests, could potentially be used for gene silencing in a variety of other animal species. Moreover, chitosan, an inexpensive, non-toxic and biodegradable polymer22, could potentially be utilized in the field for species-specific mosquito control.

<table><tbody><tr><td>0.02 ml pipetteman</td><td>Rainin</td><td>PR20</td><td>for resuspension of interfering RNA</td></tr><tr><td>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><td>0.5 ml graduated tube&amp;nbsp;</td><td>Fischer Scientific</td><td>05-408-120</td><td>for aliquoting resuspended interfering RNA</td></tr><tr><td>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><td>1.5 ml graduated tube&amp;nbsp;</td><td>Fischer Scientific</td><td>05-408-046</td><td>for preparation of interfering RNA/nanoparticles</td></tr><tr><td>1M Sodium Acetate, pH 4.5</td><td>TEKnova</td><td>S0299</td><td>to prepare sodium acetate buffer</td></tr><tr><td>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><td>Agarose Genetic Technology Grade</td><td>MP Biomedicals LLC.</td><td>800668</td><td>to coat prepared interfering RNA/nanoparticles</td></tr><tr><td>Centrifuge</td><td>Eppendorf AG</td><td>5415D</td><td>to pellet interfering RNA/nanoparticles</td></tr><tr><td>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><td>Dry Yeast</td><td>Universal Food Corp</td><td>NA</td><td>to prepare mosquito larval food with interfering RNA/nanoparticles</td></tr><tr><td>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><td>goldfish food</td><td>Wardley</td><td>Goldfish FLAKE FOOD</td><td>to prepare mosquito larval food with interfering RNA/nanoparticles</td></tr><tr><td>Heated water bath</td><td>Thermo Scientific</td><td>51221048</td><td>to heat the interfering RNA/nanoparticles at 55&lt;sup&gt;o&lt;/sup&gt;C</td></tr><tr><td>Ice</td><td>not applicable</td><td>not applicable</td><td>for thawing interfering RNA and preparation of interfering RNA/nanoparticles</td></tr><tr><td colspan="4">[header]</td></tr><tr><td>Ice bucket</td><td>Fisher Scientific</td><td>02-591-44</td><td>for storage of ice used during the procedure</td></tr><tr><td>liver powder</td><td>MP Biomedicals LLC.</td><td>900396</td><td>to feed mosquito larvae post interfering RNA/nanoparticle treatment</td></tr><tr><td>Microwave oven</td><td>A variety of vendors</td><td>not applicable</td><td>to prepare 2% agarose solution</td></tr><tr><td>petridish (100 x 15 mm)</td><td>Fischer Scientific</td><td>875713</td><td>interfering RNA/nanoparticle feeding chamber for larvae</td></tr><tr><td>pH meter</td><td>Mettler Toledo</td><td>S220</td><td>for preparation of buffers</td></tr><tr><td>Razor blade</td><td>Fischer Scientific</td><td>12-640</td><td>to divide the interfering RNA/nanoparticle pellet for feedings</td></tr><tr><td>siRNA</td><td>Thermo Scientific/Dharmacon</td><td>custom</td><td>for preparation of siRNA/nanoparticles</td></tr><tr><td>Sodium Sulfate, Anhydrous (Na&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;)</td><td>BDH/distributed by VWR</td><td>VWR BDH0302-500G</td><td>to prepare 50 mM sodium sulfate solution in which the interfering RNA will be resuspended</td></tr><tr><td>Thermometer</td><td>VWR</td><td>61066-046</td><td>to measure the water bath temperature</td></tr><tr><td>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><td>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 -80&lt;sup&gt;o&lt;/sup&gt;C</td></tr><tr><td>Vortex mixer</td><td>Fischer Scientific</td><td>02-216-108</td><td>for preparation of chitosan/interfering RNA</td></tr><tr><td>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

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

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the primary African malaria vector Anopheles gambiae and the dengue and yellow fever vector Aedes aegypti. RNA interference- (RNAi-) mediated gene silencing has been used to target genes of interest in both of these disease vector mosquito species. Here, we describe a procedure for preparation of chitosan/interfering RNA nanoparticles that are combined with food and ingested by larvae. This technically straightforward, high-throughput, and relatively inexpensive methodology, which is compatible with long double stranded RNA (dsRNA) or small interfering RNA (siRNA) molecules, has been used for the successful knockdown of a number of different genes in A. gambiae and A. aegypti larvae. Following larval feedings, knockdown, which is verified through qRT-PCR or in situ hybridization, can persist at least through the late pupal stage. This methodology may be applicable to a wide variety of mosquito and other insect species, including agricultural pests, as well as other non-model organisms. In addition to its utility in the research laboratory, in the future, chitosan, an inexpensive, non-toxic and biodegradable polymer, could potentially be utilized in the field.

A. gambiae: 
Chitosan/dsRNA nanoparticles are formed due to the electrostatic interaction between amino groups of chitosan and the phosphate groups of dsRNA. The efficiency of dsRNA incorporation into nanoparticles is usually above 90% as measured by depletion of dsRNA from the solution. Atomic force microscopy images show that chitosan-dsRNA particle size averages 140 nm in diameter, ranging from 100-200 nm (Figure 1).
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Here we describe a procedure for inhibiting gene function in disease vector mosquitoes through the use of chitosan/interfering RNA nanoparticles that are ingested by larvae.

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

The overall goal of this procedure is to design and use tussin interfering, RNA nanoparticles for gene silencing studies and mosquito larvae. This is accomplished by first preparing tussin interfering RNA nanoparticles by the combining of chitin and interfering RNA, followed by heating vortexing and centrifugation. The next goal is to mix the nanoparticles with larva food and aros to prepare food and nanoparticle containing gel pellets.The nanoparticle food is fed to the mosquito larvae and larvae feeding on the pellet is observed ultimately following verification of gene silencing by quantitative real-time polymerase chain reaction. Or in C two hybridization assays. Loss of function phenotypes can be examined.This method can help answer key questions in the vector mosquito research field. For example, we've used it to study the functions of genes during development of the olfactory system, brain midgut, and other tissues of vector importance. The main advantages of this technique are that it is high throughput, relatively inexpensive and less stressful to the organism than delivery of interfering RNA through microinjection, which requires a greater level of skill and cannot be implemented in the field.Along with postdoctoral fellow Ava Maur technician Ping Lee will be demonstrating the procedure To begin dissolve tussin in 0.1 molar sodium acetate buffer to make a 0.02%working solution. Keep the solution at room temperature before use. Then dissolved double stranded RNA or small interfering RNA, both of which are hereafter referred to as interfering RNA in 50 microliters of deionized water.Make a 100 microliter solution of 32 micrograms of nucleic acid per 100 microliters of 50 millimolar sodium sulfate. Next, add 100 microliters of tussin solution to the interfering RNA solution. Heat the mixture in a water bath at 55 degrees Celsius for one minute.Set up a control by adding 100 microliters of 50 millimolar sodium sulfate to 100 microliters of Tustin solution and follow the same procedure after heating. Mix the solutions immediately for 30 seconds by high speed vortexing at room temperature to facilitate the formation of nanoparticles centrifuge, the mixture at 13, 000 G for 10 minutes at room temperature after which app pellet should be visible. Transfer the supinate to a new 1.5 milliliter tube air dry the pellet for approximately 10 minutes at room temperature before using it.To prepare mosquito food using UV spectra photometry, measure the concentration of interfering RNA in the SNAT by using the S supernatant from the control as a blank to calculate the total amount of interfering RNA that remains in the snat. Use the difference between starting amounts of interfering RNA and amounts remaining in the supernatants to calculate the percentage of interfering RNA and trapped in the nanoparticles. This loading efficiency is normally over 90%Repeat the same procedure if more nanoparticles are needed.Use the dried nanoparticles immediately as the impact of cold storage of particles prior to use has not been evaluated. To formulate the mosquito food first, prepare a 2%agro solution in deionized water and melt the aros. Keep the melted agro solution in a 55 degree Celsius water bath before use.Next mix fish food flakes and dry yeast at a ratio of one to two. Grind the mixture to small particles with a mortar and pestle. The ground food is brownish in color.In a 1.5 milliliter tube, make six milligrams of ground food with the dry nanoparticles using a toothpick. Then add 30 microliters of 2%prem melted agros gel solution to the food nanoparticle mixture. Stir immediately and thoroughly by using a toothpick or pipet tip.The gel containing food and nanoparticles is now ready to feed the mosquito larvae to feed anomalies gambi mosquito larvae. Remove a single gel pellet from the 1.5 milliliter tube using a toothpick and cut it into four equal slices using a clean razor blade or toothpick. Then transfer 23rd in star larvae to a Petri dish containing 100 milliliters of deionized water.Feed the mosquito larvae by adding one slice of the gel pellet. Finally chopped into small pieces per Petri dish. Once a day for four days, be sure to observe larvae feeding on the pellet, which should be significantly reduced in size or completely absent by the next day after time.Mosquitoes will develop into late fourth in larvae. Record any visible phenotypic changes during the experiment. Examine the transcript levels and other phenotypic changes as discussed in the text protocol at the end of the four day period to feed eighties GTI mosquito larvae.Cut the gel pellet into four equal slices using a clean razor blade or toothpick as before. Then place 50 h synchronized 24 hour after egg hatching. First in star larvae into a Petri dish.In approximately 40 milliliters of deionized water. Feed the larvae one slice per Petri dish for four hours per day. Again, the slice should be finely chopped into smaller pieces.Then transfer larvae back to the regular larval diet of two to one ground fish food flakes and dry yeast for the rest of the day. Repeat the procedure daily throughout the four larval instar. Examine the transcript levels and other phenotypic changes as discussed in the text protocol at the desired developmental time.Points shown here is a control fed fourth instar anis gambi larvae looking normal as expected. However, the para atrophic matrix is disrupted in larvae, fed with chitin interfering, RNA, targeting chitin synthase one as evidence by dextrin blue, which has leaked into the gastric C key normal expression of orco. The obligate odorant coreceptor is observed in the antennae of control fed fourth instar eighties aegypti larvae.In contrast, Orco expression is absent in animals fed with tussin interfering, RNA, targeting the single-minded gene in comparison to control fed animals. Single-minded knockdown animals have defective odorant tracking behavior. After watching this video, it should have a good understanding of how to use Keon interfering, RNA nanoparticles to silence genes during vector mosquito development.This technique which could potentially be extended to the study of adult mosquitoes or other emerging model organisms has paved the way for researchers to explore the functions of genes identified in disease vector, mosquito genome projects.

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

Research

JoVE Journal

Biology

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite.  The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Protocol for RNAi Assays in Adult Mosquitoes (A. gambiae)

In this protocol we combine RNAi-mediated gene silencing with an in vivo diuresis assay to study the effects knockdown of genes of interest has on mosquito fluid excretion.

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

RNA interference (RNAi) is an extremely valuable tool for uncovering gene function. However, the ability to target genes using RNAi during pre-adult stages is limited in the major human malaria vector Anopheles gambiae. We describe an RNAi protocol to reduce gene function via direct injection during pupal development.

RNAi Trigger Delivery into Anopheles gambiae Pupae

We report using an artificial intronic small RNA expression strategy to develop a non-defective recombinant Aedes aegypti densovirus (AaeDV) in vivo delivery system. A detailed procedure for the construction, packaging, and quantitative analysis of the rAaeDV vectors as well as for larval infection is described.

Use of a Recombinant Mosquito Densovirus As a Gene Delivery Vector for the Functional Analysis of Genes in Mosquito Larvae

This article demonstrates novel techniques developed for oral delivery of double-stranded RNA (dsRNA) through the vascular tissues of plants for RNA interference (RNAi) in phloem sap feeding insects.

Double-stranded RNA Oral Delivery Methods to Induce RNA Interference in Phloem and Plant-sap-feeding Hemipteran Insects

This protocol describes a microinjection methodology that we have standardized and used for several years to deliver specific quantities of nucleic acids directly to the hemolymph of mosquitoes and house flies. This protocol results in minimal injection mortality and allows dose correlated measurements of fecundity.

Reproducible dsRNA Microinjection and Oviposition Bioassay in Mosquitoes and House Flies

The oral administration of dsRNA produced by bacteria, a delivery method for RNA interference (RNAi) that is routinely used in Caenorhabditis elegans, was successfully applied here to adult mosquitoes. Our method allows for robust reverse genetics studies and transmission-blocking vector studies without the use of injection.

Effective Oral RNA Interference (RNAi) Administration to Adult Anopheles gambiae Mosquitoes

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

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

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

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

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.

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

Learn chitosan/interfering RNA nanoparticle gene silencing in disease vector mosquito larvae. This method enables functional studies of genes in Anopheles gambiae and Aedes aegypti using oral delivery.

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

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