ミツバチにおけるRNAiによる二重遺伝子ノックダウンと味覚知覚測定（

This video protocol demonstrates novel techniques to simultaneously knock down two genes in adult honeybees and then conduct a probos extension response or PER assay to measure the effect of the double gene knockdown. On honeybee gustatory perception. This is accomplished by first synthesizing double stranded RNAs, targeting the genes for three proteins, vitelli, genin, ultras, spherical, and green fluorescence protein.

Since the gene encoding GFP is not found in the honeybee genome, the GFP double stranded RNA serves as a negative control. The second step is to inject double stranded RNA into honeybees using four treatment strategies. Injection with GFP double stranded, RNA alone, a single injection with a VG and USP double stranded, RNA mixture injection with vg double stranded RNA on the first day, followed by injection with USP double stranded RNA on the second day, and injection with USP double stranded RNA on the first day, followed by injection with VG doublet stranded RNA on the second day after the injections marked the bees with color coded paint dots corresponding to the treatments and returned them to a hive.

Six days later, VG and USP gene knockdown in the treated bees is validated by realtime PCR and the effect of double VG and USP knockdown on gustatory perception is determined by the PER assay. Ultimately double knockdown bees are shown to have significant reductions in the expression of VG and USP genes and significant increases in gustatory perception to sucrose. Our interference is a powerful tool we can use to reveal gene functions.

It's typically used to target a single gene. However, genes are part of complex regulatory network. One way we can better understand any biological process is to learn how gene interact with gene network.

To do this, we have to manipulate multiple genes at the same time. Until now, we have only been able to surprise one gene at a time in insects here for the first time, we want to show you how to knock down two Gs in honeybees instead of one. The SCUs extension reflex or PER is a very useful technique for measuring gustatory perception, which is a behavioral predictor for honeybee social behavior.

For example, nest bees with high gustatory perception, behaviorally mature fast, forge early in life, and prefer to collect pollen. To begin this procedure, design primers of double stranded RNAs targeting the genes for knockdown using the free software primer.Three. In this experiment, the two genes targeted for knockdown are vitelli, genin, or VG and ultras spherical or USP.

In addition to VG and USP design, primers of double stranded RNA targeting a control gene encoding green fluorescence protein, or GFP, which is not in the honeybee genome. Subsequently double stranded. RNA synthesis for VG USP and GFP is accomplished by in vitro transcription using the rib max T seven RNA production system from Promega.

After purifying the double stranded RNAs using standard procedures dissolve each double stranded RNA pellet in nuclease free water in order to ensure the efficacy of gene downregulation, the double stranded RNA concentration should be around nine to 10 micrograms per microliter. To prepare the bees for injection chill, newly emerged bees in a four degree Celsius refrigerator for one to two minutes. Once the bees are immobilized, mount three to four bees in parallel on Petri dishes full of solid wax with insect pins crossed between their abdomens and thoracis.

Chill the bees again in a four degree Celsius refrigerator for about one to two minutes. Ensure the bees are completely immobile. The bees should look loose, not curled or contorted.

Complete immobilization is important as any movement could increase the size of the wound and mortality. Bees that are curled or contorted have been chilled for too long and should not be used because of high mortality. Put a disposable 30 gauge needle on a Hamilton micro syringe.

Fill the syringe with three microliters of double stranded RNA and to make sure there is no air bubble in the syringe. If a single injection strategy is used, the syringe is filled with a mixture of the double stranded RNAs of the two genes. If a two day injection strategy is used, the syringe is filled with one of the two double stranded RNAs.

Insert the needle into a side of the abdomen. To avoid damaging internal organs, press the syringe plunger slowly to allow the double stranded RNA to be absorbed. Since double stranded RNA is very viscous, it takes two to three seconds to completely expel it from the syringe.

After completely pushing down the plunger, leave the needle in the wound for four to five seconds. Observe bees for three to five seconds after injections. If a hemo lymph droplet leaks from the wound, discard the bee, mark the thoracis of the bees with different colors corresponding to different treatments.

At this point, if the single injection strategy is being used, observe the bees for one hour in a container with honey supplied, then place them back into a colony. However, if the two day injection strategy is used and only the first double stranded RNA has been injected, place the bees in a cylinder mesh cage with honey supplied on the side. Keep the bees in an incubator at 34 degrees Celsius and 80%humidity overnight on the following day.

Inject the same bees with the second double stranded RNA, using the same procedure as for the first double stranded RNA. Let the bees recover at room temperature for one hour in a container with honey supplied, and then introduce them into a colony. Six days the injections collect treated bees from the colony without using a smoker to open the hive.

Smoke is not used because it could affect the gustatory perception of bees, which will be tested in this procedure. Collect bees in metal mesh cylinder cages using soft forceps with no more than three bees per cage to mount bees. For the PER assay.

Chill the bees at four degrees Celsius until they are completely immobilized. Mount each bee vertically into a plastic tube, specially designed for PER. Use tape to keep the abdomen inside the tube and keep the head sticking out of the tube.

Put the tubes on a rack with two rows of small plastic columns. One row is numbered so the mounted bees can be assigned an ID number. Put the racks with the treated bees into an incubator at 34 degrees Celsius and 80%humidity for two hours while the bees are incubating.

Prepare a series of sucrose. Water solutions. Load the solutions into syringes with 15 to 20 gauge needles for the PER assay.

The bees are tested in groups. First touch each bee's antenna with a droplet of 0%solution, which is water after that test with 0.1%0.3%1%3%10%and 30%sucrose. Water solutions allowing at least two minutes for the interval between each solution.

It usually takes around two minutes to test 20 bees using one concentration of sucrose. Therefore, there should always be more than 20 bees in each group. If a bee fully extends its probos, a positive response is counted.Real-time.

PCR analysis confirmed that both the single injection and two day injection strategies significantly reduced Fiji and USP transcript levels in honeybees. Six days after the double stranded RNA was injected in these graphs, the RQ indicates relative quantification level of the target gene transcripts and the different letters above the bars denote significant differences between the treatment groups. Post talk analysis revealed that the suppression of USP transcript by using the single injection with double stranded RNA mixture and the two day injection with VG first and USP second was significantly lower than the two day injection with USP first and VG second.

This indicates that VG may be a primary regulator in the VG and juvenile hormone regulatory loop. GUA to response scores or GRS for the treated bees were calculated from results of the probos extension response or PER assay as shown here. The GRS in the bees that were injected with VG and USP double stranded RNA mixture was significantly higher than the GRS in the GFP control bees indicating the double knockdown bees were more sensitive to sucrose.

The different letters above the bars denote significant differences between the treatment groups. Shown here is a possible regulatory network between vi genin ultras, spherical, and juvenile hormone revealed by simultaneously suppressing VG and USP genes. While a USP single knockdown does not induce any change in juvenile hormone, a double knockdown of VG and USP causes a greater increase in juvenile hormone than a VG single knockdown.

This suggests that vitelli not only inhibits juvenile hormone production, but inhibits a feedback response of juvenile hormone to USP knockdown. In double knockdowns, the dramatic increase of juvenile hormone production results from a reduced inhibition from VI genin caused by VG knockdown and a compensatory response to a reduced juvenile hormone transduction caused by USP knockdown. A more detailed discussion of this regulatory network is found in the accompanying protocol text.

By using double gene knockdown approach, we have revealed a detailed relationship between vital genic, ultras, spherical, and juvenile hormone, which we would not have discovered by suppressing only one gene. Now, by using this technique, researchers can explore the interrelationship between genes within regulatory networks. PR is a standard method used to measure gustatory perception In honeybees, however, it is very sensitive to handling, so one must be very careful when keeping consistencies between the environment and handling of the animals.

In addition, if the essay's gonna be performed over several days, it's a good idea to keep a short timeline and keep an eye to make sure the weather and the environment doesn't change.