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>

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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 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

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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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

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 ...

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

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Biology

14.2K Views.  Kansas State University. 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.

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.

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

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.

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.

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

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

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

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

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

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

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.

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.

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.

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.

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.

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

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, most notably developmental arrest and reduced metabolism. Global gene expression profiling via RNA-Seq can provide important insights into the transcriptional mechanisms of photoperiodic diapause. The Asian tiger mosquito, Aedes albopictus, is an outstanding organism for studying the transcriptional bases of diapause due to its ease of rearing, easily induced diapause, and the genomic resources available. This manuscript presents a general experimental workflow for identifying diapause-induced transcriptional differences in A. albopictus. Rearing techniques, conditions necessary to induce diapause and non-diapause development, methods to estimate percent diapause in a population, and RNA extraction and integrity assessment for mosquitoes are documented. A workflow to process RNA-Seq data from Illumina sequencers culminates in a list of differentially expressed genes. The representative results demonstrate that this protocol can be used to effectively identify genes differentially regulated at the transcriptional level in A. albopictus due to photoperiodic differences. With modest adjustments, this workflow can be readily adapted to study the transcriptional bases of diapause or other important life history traits in other mosquitoes.

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

RNA-Seq analyses are becoming increasingly important for identifying the molecular underpinnings of adaptive traits in non-model organisms. Here, a protocol to identify differentially expressed genes between diapause and non-diapause Aedes albopictus mosquitoes is described, from mosquito rearing, to RNA sequencing and bioinformatics analyses of RNA-Seq data.

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, ...