隠すビートルにノックダウン飼育し、二本鎖RNA媒介遺伝子、

The overall goal of this procedure is to explore gene function in Dermestes maculatus, a new insect model system, which represents the diverse clade of beetles, and facilitates the study of basic and applied research questions. This method can help answer key questions in the fields of developmental biology, evolutionary biology and beyond, by making it possible to test gene function in Dermestes embryos and adults. The main advantage of this technique is that RNA interference can be used to target any gene, based only on sequence information, even in the absence of a full genome sequence.

Methods introduced here not only provide an approach to study gene function in Dermestes. They can also be applied to develop new insect model systems, and potentially to control insect pests. To prepare a rearing cage for a D.maculatus, first spread a thin layer of wood shavings into a medium sized insect cage.

Place a section of styrofoam into the cage to allow larvae an environment, in which to pupate. Add twenty to fifty adult beetles or late instar larvae to the cage. Place wet cat food into a Petri dish or weigh boat, as a food source.

Cover the cage with a mesh cloth and place it into an incubator. Before starting embryo collection, first check the cage carefully for any embryos present and remove them. Once the cage is clear, place a stretched cotton ball into the cage and incubate the colony at 30 degrees Celsius.

After the appropriate time window, collect the cotton ball, and remove any adults present, and place them back into the cage. Over a piece of black construction paper, pinch the cotton ball gently, and tear it slowly into thin filament, so that any eggs present fall onto the black paper. Take a standard Petri dish lid, and use a piece of black filter paper to cover the inside.

Stick a piece of double-sided tape along the edge of a standard microscope slide. Place the microscope slide onto the black filter paper in the Petri dish lid. Taking the paper containing the collected embryos, gently tap to transfer the embryos to the Petri dish lid.

Using a paintbrush, align the embryos on the tape perpendicular to the slide, with the anterior or posterior end towards the edge. To begin RNAi, first take up 2-4 microliters of dsRNA into a 20 microliter micro loader pipette tip. Insert the tip into a pre-pulled glass capillary, referred to as the needle, and gently pipette the solution into the needle.

Fix the needle into a glass capillary holder and then assemble the capillary holder into the micromanipulator. Next, carefully transfer the slide with embryos attached onto the stage of the dissecting microscope. Move the slide to bring one embryo into the center of the field and focus the microscope.

Whilst looking into the dissecting scope, position the tip of the capillary with the micromanipulator. Bring the tip close to the end of the first embryo in the row. Switch on the nitrogen or air supply, and then set the solenoid input selector switch to vacuum.

Adjust the eject pressure regulator to 10-15 psi, depending on the apparatus. Open the capillary, and the colored dsRNA solution will fill the tip, ready for injection. Move the capillary tip forward, and gently puncture the embryo.

Under ideal pressure settings, no embryonic fluid will flow into the capillary. Step on the foot switch to eject dsRNA into the embryo, until the appropriate amount is dispensed. Leave the tip inside the embryo for around two seconds, and then carefully remove it.

Once all of the embryos have been injected, place the slide into a Petri dish. Add a wet cotton ball to the dish, taking care not to let it come into contact with the slide. Cover the Petri dish and wrap it with sealing film.

Label it with the dsRNA type, concentration, date, time and number of injected embryos. Finally, place the dish into an incubator at 30 degrees Celsius until the embryos hatch. To perform pRNAi, first collect pupae from the colony.

Sort male and female pupae into two separate dishes, and place these into a 30 degree Celcius incubator. Check the plates daily for eclosed beetles, and transfer these individuals to two separate Petri dishes for male and female adults. Feed these adults and maintain the plates every other day, until enough adults have eclosed to begin injection.

Once the adult pool is complete, anesthetize a female beetle, and hold the individual ventral side up with one hand, holding the syringe in the other. Gently penetrate the segmacoria membrane between sternites 2 and 3. Check the syringe scale and slowly dispense around two microliters per female.

After injection, hold the needle steady for at least 5 seconds and then carefully withdraw it. Transfer injected females to a Petri dish with cat food. Label the dish appropriately.

Place the dish into a 30 degrees Celcius incubator. After 24 hours, transfer the injected females to a mini cage, and add an equal number of the uninjected young males. Label the cage, and add cat food.

Place the cage into a 30 degree Celsius incubator, to allow mating. The resultant offspring can then be examined to determine the effects of the pRNAi. This image shows an offspring of a pRNAi individual injected with DS GFP, green fluorescent protein as a control.

Here, as with wild-type individuals, the offspring hatched with one pigmented stripe per segment. Neighboring pigmented stripes were separated by a non-pigmented gap. Following paired pRNAi, affected offspring hatched with fused neighboring pigmented stripes, indicating defective segmental boundaries.

Phenotypic severity was variable, but fusions consistently appeared at certain boundary regions, indicating pair rule like defects. Similarly, cuticle phenotypes of unhatched embryos after paired knockdown showed loss of abdominal segments, as well as shortened body length. Staining of early embryos with engrailed antibody to examine molecular defects showed stripes of expression of equal intensity in each segment in control individuals.

In contrast, reduced expression was detected in alternate stripes in offspring of Dmac paired dsRNA injected females. This pattern of reduced expression is consistent with the defective cuticle pattern observed in the affected hatched larvae. Here, for the first time, we present our protocol for knocking down a gene of interest, during the early embryonic development in Dermestes maculatus, using RNA interference.

While attempting these procedures, it is important to remember to use embryos or female adults at the appropriate stage. Once mastered, injecting about 100 embryos or 10 female adults can be done within half an hour. Following this procedure, other methods, like embryo staining or quantitative PCR can be performed in order to answer additional questions about gene regulatory mechanisms.

After watching this video, you should have a good understanding of how to set up a Dermestes lab colony, and perform both embryonic and parental RNA interference in this beetle. Currently, only a small number of insect models are available for functional studies. The techniques presented here pave the way for researchers to use Dermestes as a model system, expanding the scope of insects amenable to molecular genetic analysis.