The authors acknowledge financial support from the National Key Research and Development Program of China (2016YFC1200500 to Xiao-Guang Chen), the National Natural Science Foundation of China (81672054 and 81371846), the Research Team Programme of the Natural Science Foundation of Guangdong (2014A030312016), and the Scientific and Technological Programme of Guangzhou (201508020263). We gratefully acknowledge Professor Jonathan Carlson (Colorado State University) for kindly providing the pUCA and p7NS1-GFP plasmids and for critically reading this manuscript.

All protocols were approved by the Ethical Committee of Southern Medical University.
1. Designing an Artificial Intron
NOTE: The artificial intron used in this work is 65 bp in length, and the sequence is 5&#39;-GTAAGAGTCGATCGACGCGTTACTAACTGGTACCTCTTCTTTTTTTTTTGATATCCTGCAG-3&#39;.
<ol>
	<li>Place the HpaI restriction site on both ends of artificial intron so that HpaI digestion can release the intron from plasmids (see Figure...

<ol>
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The authors declare that they have no competing financial interests.

It is important to overcome two of the key barriers that limit rAaeDV construction. The first is the production of defective recombinant virus. It has been reported that MDV can be used as a vector to express appropriately sized foreign genes, such as scorpion insect-specific neurotoxins20 and the GFP protein; however, regardless of the construction strategy, once the ORFs of MDV are inserted or replaced with exogenous genes, virions cannot form independently unless a helper plasmid is cotransfected to supply the missing...

Mosquito-borne diseases such as malaria, dengue fever, zika fever, and yellow fever, are major international public health problems that continue to account for a significant fraction of the global infectious disease burden1,2. Conventional insecticides, which have been used in response to vectors, are a major component of sustainable integrated mosquito control strategy for the prevention of mosquito-borne diseases. However, such strategies have proven to be relatively ineffective or undesirable due to the associated negative environmental impacts as well as the evolution of resistance in mosquito populations3,4. For these reasons, there is an urgent need for alternative methods of mosquito control, and the use of transgenic methods to produce sterile male mosquitoes and the release of pathogen-resistant mosquitoes have arisen as promising new control strategies. To develop effective new control methods, such as safe and effective approaches for in vivo gene delivery, the comprehensive analysis of mosquito gene function is required.
Direct microinjection of plasmid DNA, double stranded RNA (dsRNA) or small interfering RNA (siRNA) is the most commonly used in vivo gene delivery method in mosquitoes. In fact, the production of transgenic strains of mosquitoes still rely on a process of embryo microinjection5,6,7. However, microinjection has several limitations.First, this technique is technically demanding and involves complicated procedures. Second, injection causes a physical insult to the embryo, larvae, pupae, and adult, which directly affects the viability of the target organism. Third, it is difficult to immobilize mosquito larvae for microinjection because most live in an aquatic habitat and possess a characteristic wriggling movement. Fourth, the size of 1st-2nd instar larvae is 10- to 20-fold smaller than that of 4th instar and older larvae, and the cuticles of the former are more delicate. These features make it difficult to manipulate 1st-2nd instar larvae compared to those in older stages. Combined, these factors contribute to a reduced post-injection survival rate for larvae (approximately 5%) compared to adults8. Viral-based delivery systems have been developed to overcome the associated extracellular and intracellular barriers. These systems have the advantages of easy manipulation, high transduction efficiency, long-term and robust levels of expression, and the ability to produce persistent effects in vivo. Therefore, gene delivery systems utilizing retroviruses, lentiviruses, and adenoviruses have been widely used inmammalian cell lines and model species. The Sindbis viral expression system had been previously used to analyze the gene function in the adult mosquito; besides the biosafety concerns, however, the injection is still a necessary process for viral infection9. Although, the oral delivery by soaking larvae in the dsRNA solution has been reported previously as a feasible delivery method, it is unsuitable for small RNA function analysis10. So, effective viral delivery methods must still be developed for mosquitoes.
Mosquito densoviruses (MDVs) are part of the Densovirinae subfamily of Parvoviridae, and all but one fall within the genus Brevidensovirus11. MDV virions are non-enveloped and consist of a single-stranded DNA (ssDNA) genome and an icosahedral capsid (20 nm in diameter). The viral genome is approximately 4 kb in size and is replicated within the nuclei of host cells. MDVs are relatively stable in the environment and show a narrow host range with high specificity for mosquitoes. These viruses have the potential to spread and persist naturally in mosquito populations and can invade almost all organs and tissues of these insects, including the midgut, Malpighian tubules, fat body, musculature, neurons, and salivary glands12.
Intact MDV genomes can be subcloned into plasmid vectors to produce plasmid-based infectious clones; when these clones are delivered into mosquito cells, the viral genome is extracted from the plasmid vector, and infectious viral particles are produced. Because MDV has a small ssDNA genome, infectious clones are easily constructed and the viral genome can be easily manipulated11,13. These characteristics make MDV a valuable agent for examining mosquito biology. However, because nearly all of the viral genome sequence is essential for viral proliferation, the creation of recombinant virus through the replacement or insertion of foreign genes causes a loss in viral packaging and/or replication abilities, which creates a barrier for the development of MDVs as gene delivery vectors. Herein, we report using an artificial intronic small-RNA expression strategy to develop a non-defective rAaeDV in vivo RNA delivery system that has the advantages of easy plasmid construction and the maintenance of a functional virus that can produce stable and long-term expression in host cells without the need for wild-type virus. Additionally, this method allows for the easy transduction of larvae.
The protocols for the following steps are described in this study: 1) design of rAaeDVs encoding an intronic small-RNA expression cassette, 2) production of recombinant virus particles using the C6/36 packaging cell line, 3) quantitative analysis of cell-free rAaeDV genome copy numbers, and 4) infection of Ae. albopictus larvae by direct introduction of virus into the water body of the larval habitat. Overall, this work demonstrated that specific small RNAs or target genes can be overexpressed or knocked down in mosquito larvae using the developed MDV delivery system.

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			<td height="24" style="width:401px;height:24px;">Centrifuge machine</td>
			<td style="width:215px;">Thermo Scientific</td>
			<td style="width:119px;">75004260</td>
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			<td height="21" style="width:401px;height:21px;">Centrifuge System</td>
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			<td height="21" style="width:401px;height:21px;">GeneJET Gel Extraction Kit</td>
			<td style="width:215px;">Thermo Scientific</td>
			<td style="width:119px;">K0691</td>
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			<td height="21" style="width:401px;height:21px;">E.Z.N.A. Plasmid Midi Kit</td>
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			<td height="21" style="width:401px;height:21px;">Purified Agar</td>
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			<td style="width:119px;">LP0028A</td>
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			<td height="21" style="width:401px;height:21px;">FastAP Thermosensitive Alkaline Phosphatase (1 U/&#181;L)</td>
			<td style="width:215px;">Thermo Scientific</td>
			<td style="width:119px;">EF0651</td>
			<td></td>
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			<td height="21" style="width:401px;height:21px;">FastDigest HpaI</td>
			<td style="width:215px;">Thermo Scientific</td>
			<td style="width:119px;">FD1034</td>
			<td></td>
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			<td height="21" style="width:401px;height:21px;">FastDigest XbaI</td>
			<td style="width:215px;">Thermo Scientific</td>
			<td style="width:119px;">FD0684</td>
			<td></td>
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			<td height="21" style="width:401px;height:21px;">T4 DNA Ligase (5 U/&#181;L)</td>
			<td style="width:215px;">Thermo Scientific</td>
			<td style="width:119px;">EL0011</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="width:401px;height:21px;">TransStbl3 Chemically Competent Cell</td>
			<td style="width:215px;">Beijing Transgen Biotech</td>
			<td style="width:119px;">CD521-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:401px;height:21px;">Ampicillin&#160;</td>
			<td style="width:215px;">Beijing Tiangen Biotech</td>
			<td style="width:119px;">RT501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:401px;height:21px;">Roswell Park Memorial Institute (RPMI) 1640 medium</td>
			<td style="width:215px;">Gibco&#65292; Life Technologies</td>
			<td style="width:119px;">&#160;21875109</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:401px;height:21px;">Fetal Bovine Serum&#65288; Australia Origin&#65289;</td>
			<td style="width:215px;">Gibco&#65292; Life Technologies</td>
			<td style="width:119px;">10099141</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:401px;height:21px;">penicillin/streptomycin</td>
			<td style="width:215px;">Gibco&#65292; Life Technologies</td>
			<td style="width:119px;">15070063</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="width:401px;height:23px;">Lipofectamine 2000 Transfection Reagent</td>
			<td style="width:215px;">&#160;Invitrogen&#65292;Life Technologies</td>
			<td style="width:119px;">11668019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:401px;height:21px;">Proteinase K</td>
			<td style="width:215px;">Promega</td>
			<td>MC5008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:401px;height:21px;">DNase I (RNase-free)</td>
			<td style="width:215px;">&#160;Invitrogen&#65292;Life Technologies</td>
			<td style="width:119px;">AM2222</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:401px;height:21px;">SuperReal PreMix Plus (SYBR Green)</td>
			<td style="width:215px;">Beijing Tiangen Biotech</td>
			<td style="width:119px;">FP205</td>
			<td></td>
		</tr>
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use of a recombinant mosquito densovirus as a gene delivery vector for the functional analysis of genes in mosquito larvae

In vivo microinjection is the most commonly used gene transfer technique for analyzing the gene functions in individual mosquitoes. However, this method requires a more technically demanding operation and involves complicated procedures, especially when used in larvae due to their small size, relatively thin and fragile cuticle, and high mortality, which limit its application. In contrast, viral vectors for gene delivery have been developed to surmount extracellular and intracellular barriers. These systems have the advantages of easy manipulation, high gene transduction efficiency, long-term maintenance of gene expression, and the ability to produce persistent effects in vivo. Mosquito densoviruses (MDVs) are mosquito-specific, small single-stranded DNA viruses that can effectively deliver foreign nucleic acids into mosquito cells; however, the replacement or insertion of foreign genes to create recombinant viruses typically causes a loss of packaging and/or replication abilities, which is a barrier to the development of these viruses as delivery vectors.
Herein, we report using an artificial intronic small-RNA expression strategy to develop a non-defective recombinant Aedes aegypti densovirus (AaeDV) in vivo delivery system. Detailed procedures for the construction, packaging and quantitative analysis of the rAaeDV vectors, and for larval infection are described.
This study demonstrates, for the first time, the feasibility of developing a non-defective recombinant MDV micro RNA (miRNA) expression system, and thus providing a powerful tool for the functional analysis of genes in mosquito and establishing a basis for the application of viral paratransgenesis for controlling mosquito-borne diseases. We demonstrated that Aedes albopictus 1st instar larvae could be easily and effectively infected by introducing the virus into the water body of the larvae breeding site and that the developed rAaeDVs could be used to overexpress or knock down the expression of a specific target gene in larvae, providing a tool for the functional analysis of mosquito genes.

Strategies for rAaeDV construction&#160; 
A defective rAaeDV vector was generated to express the DsRed gene in mosquito larvae. The resulting plasmid contained a NS1-DsRed fusion protein cassette with the VP protein deleted (Figure 1A). rAaeDV plasmids containing expression constructs for miRNA, miRNA sponge, shRNA and amiRNA were designed (shown in Figure 1B). For example, ...

Watch this Scientific Journal Video about Use of a Recombinant Mosquito Densovirus As a Gene Delivery Vector for the Functional Analysis of Genes in Mosquito Larvae at JoVE.com

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

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.

In vivo microinjection is the most commonly used gene transfer technique for analyzing the gene functions in individual mosquitoes. However, this method ...

The overall goal of this procedure is to analyze the gene function of mosquito larvae, using recombinant mosquito densovirus as a gene delivery. This method can help answer key questions in viral-based delivery systems, such as how to overcome the associated extracellular and intracellular barriers. The main advantages of these techniques are easy manipulation, high transduction efficiency, long-term and robust use of expressions, and the ability to produce persistent effects in vivo.This work demonstrated that specific small RNAs or target genes can be overexpressed or knocked down in mosquito larvae, using the developed densovirus delivery system. The implications of this technique extend the applications of the described expression strategy to investigate mosquito biology because this method allows for easy transductions of larvae. Demonstrating the procedure will be Pei-Wen Liu, a doctoral candidate from our laboratory.Start by ordering the chemical synthesis of full-length cDNA sequences for the precursor miRNAs, miRNA sponges, and shRNA, as described in the accompanying text protocol. After receiving the ordered DNA, spin the tubes containing DNA blocks to ensure that all of the dried DNA is at the bottom of the tube. Then, resuspend the DNA in DNase-free water to achieve a final concentration of 10 nanograms per microliter.Add six microliters of XbaI to digest plasmid backbone and cDNA sequences, and place the reaction at 37 degrees Celsius for one hour. Then, dephosphorylate the five-prime ends of the linearized plasmid backbone with calf-intestinal alkaline phosphatase, according to the manufacturer's instructions. For efficient packaging, the genome size cannot exceed 4, 400 nucleotide, representing 110%of the length of the wild-type mosquito densovirus genome, or intronic microRNA expression consist of no more than 400 nucleotide can be introduced into the Aedes aegypti genome.Next, heat inactivate the alkaline phosphatase at 65 degrees Celsius. Then, run the DNA on a 1%agarose gel, and cut the linearized backbone. Extract the DNA from the gel slice, using a gel extraction kit, following the manufacturer's instructions.Once extracted, ligate the DNA fragment into the backbone, using T4 DNA ligase. Then, use heat shock to transform the products of the ligation reaction into competent DH5-alpha E.coli cells. Plate the transformed bacteria on an LB agar plate containing 100 micrograms per milliliter of ampicillin.The next day, pick a single colony from the plate, and create a starter culture, using three milliliters enriched medium containing 100 micrograms per milliliter of ampicillin. Finally, extract the plasmid DNA from the bacterial culture, using a miniprep kit, following the manufacturer's instructions. Send a sample of the DNA to a DNA sequencing company for verification.Ligate pre-miRNA or shRNA into the HpaI site of the AaeDV NS1 coding region in the pUCA plasmid backbone, using the methods that were shown for the artificial intronic small-RNA expression cassette. In place of using DH5-alpha cells, transform the DNA into Stbl3 competent E.coli cells. Then, plate the transformed cells onto an LB agar plate containing 100 micrograms per milliliter of ampicillin, and make a starter culture from a positive clone.Following this, extract the rAaeDV plasmid from the bacterial cells, using a maxiprep kit, following the manufacturer's instructions. On day zero, split 90 to 95%confluent flasks of C6/36 cells one to two into 10 T-25 culture flasks. Incubate the flasks for 18 to 20 hours at 28 degrees Celsius until they again reach 90 to 95%confluence.On day one, transfect the cells with the generated rAaeDV plasmids. To accomplish this, dilute 10 micrograms of DNA into 600 microliters of RPMI-1640 medium, and mix the solution gently. Meanwhile, prepare 600 microliters of RPMI-1640 medium and 25 microliters of transfection reagent per transfection.Incubate the cells with the DNA for five to 10 minutes at room temperature. Then, add 625 microliters of the RPMI-1640 transfection reagent mixture to the plasmid DNA mixture, and incubate this mixture for 20 to 30 minutes at room temperature. Following incubation, wash the cells twice with RPMI-1640 medium, and then add the transfection mixture to each of the T-25 culture flasks.Incubate the cells in the transfection mixture for six hours at 28 degrees Celsius. After six hours, remove the transfection media, wash the cells twice with five milliliters of RPMI-1640 medium, and then add fresh RPMI-1640 medium containing 10%FBS. Five days after transfection, use a five-milliliter glass dropper to dislodge and suspend the cells in the dishes by pipetting up and down with the culture medium.Transfer the cell suspensions from each flask into their own sterile 50-milliliter tube. To harvest a high threshold whereas it is necessary to maintain the cells for five days after transfection. Freeze the individual tubes at negative 80 degrees Celsius or in a dry ice ethanol bath for 30 minutes.Then, quick thaw them at 37 degrees Celsius, and vortex the cell suspension for one minute. Next, collect the supernatant from the centrifuged sample, and discard the pellet. Then, pass the supernatant through a 0.22 micrometer disposable syringe filter.Aliquot and store the final purified virion stocks at negative 80 degrees Celsius. Wash 100 first-instar A.albopictus larvae three times, using deionized water. And then, transfer the larvae into a beaker containing 95 milliliters of deionized water.Add five milliliters of the rAaeDV stocks, and incubate the larvae for 24 hours at 28 degrees Celsius. For success for larva infection, it is essential for larvae to be contained with infection virus particles for 24 to 36 hour to ensure high infection ratios. Following the incubation, wash the larvae three times using deionized water, and then transfer the larvae back to the plates.Feed the larvae regularly. Monitor the level of target gene expression in the larvae, using quantitative PCR or western blot, using standard techniques. In this example, 100 first-instar larvae were analyzed for their endogenous miRNA expression after infection with the recombinant virus containing the aal-let-7 expression cassette showing overexpression.The efficiency of the anti-let-7 construct is shown on the right, highlighting its knockdown capabilities. In order to monitor the V-ATPase mRNA knockdown efficiency of the rAaeDV vectors, a total of 100 first-instar larvae were treated with equal amounts of rAaeDV of varying types by adding the virus into the water body of the larval habitat. The V-ATPase mRNA levels were substantially reduced by an miRNA expressing rAaeDV and by intronic shRNA expressing rAaeDV.Our results validate an intronic expression strategy that offers a new paradigm, which can overcome the limitations of mosquito densoviruses with defective genome, to enable efficient delivery of foreign genes into mosquitoes. While attempting this procedure, it is important to remember that an intronic microRNA expression cassette of no more than 400 nt can be introduced into the Aedes aegypti densovirus genome. After its development, this technique paved the way for research in productions of non-defective recombinant mosquito densovirus that can overexpress or downregulate microRNAs and the target genes to explore functional analysis of mosquito genes.After watching this video, you should have good understanding of how to use developed mosquito densovirus delivery system to analyze the gene functions in mosquito larvae.

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Research

JoVE Journal

Developmental Biology

6.6K Görüntüleme.  Southern Medical University. The overall goal of this procedure is to analyze the gene function of mosquito larvae, using recombinant mosquito densovirus as a gene delivery. This method can help answer key questions in viral-based delivery systems, such as how to overcome the associated extracellular and intracellular barriers. The main advantages of these techniques are easy manipulation, high transduction efficiency, long-term and robust use of expressions, and the ability to produce persistent effects in vivo.T...