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

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.