蚊幼虫における遺伝子の機能解析の遺伝子配達ベクトルとして組換え蚊 Densovirus を使用

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.