The overall goal of this procedure is to isolate extracellular fluid to answer questions of pseudo fluid, makeup and function, such as questions involving RNA eye signaling. This is accomplished by first feeding worms, bacteria expressing DS RNA, which results in an easily scored RNAi phenotype. Next, the worms are allowed to disseminate RNAi silencing signals throughout the body.
Then the total volume of available extracellular fluid is increased. Finally, a microinjection needle is used to remove the extracellular fluid and injected into an RNAi naive worm. Ultimately, the presence of RNAi silencing signals can be shown through the scoring of progeny of injected worms.
Through this method, we can analyze the composition of the extracellular fluid to provide insight not only into systemic RNAi, but a variety of intercellular signaling pathways. Generally, individuals new to this technique will struggle because of the speed with which one must work. To prepare donor worms using clear one E 1745 streak out bacteria expressing DS RNA for single colonies on LB plus CARIL plates, PAL one DS RNA producing bacteria are used for this demonstration.
Incubate the plates overnight at 37 degrees Celsius the following day. Pick a single colony to inoculate a three milliliter overnight culture in lb supplemented with carbon penicillin pipette 15 microliters of overnight culture onto 35 millimeter NGM plates. Supplemented with 25 micrograms per milliliter of carbonic and one millimolar IPTG.
Make sure to plate the bacteria off center of the plate. Allow the culture to dry. Next pipette 10 microliters of freshly made bleach solution on the seated plate.
Be sure to place the bleach droplet away from the bacterial spot to obtain a small, clean, synchronized population of clear. One E 1745 animals for reverse microinjection. Pick two to 10 grave adults and place them into the bleach drop.
The adults will dissolve leaving clean, partially developmentally staged embryos. These embryos will then hatch and the larvae will crawl to the food. Incubate the embryos for four days at 20 degrees Celsius.
Then shift the plates to 25 degrees Celsius for four hours to induce swelling. Pick worms and transfer them to an unseated NGM plate and allow time to clear off bacteria from the cuticle. Then move L four young adult recipient worms to an unseated NGM plate and allow time to clear off bacteria from the cuticle.
After preparing an overnight liquid culture of OP 50 N lb pipette 15 microliters onto 35 millimeter NGM plates. Make sure to plate the bacteria off center of the plate. Allow them to dry pipette 10 microliters of freshly prepared bleach solution on the seeded plate away from the bacterial spot.
Pick two to 10 GR N two adults and transfer them into the bleach droplet incubate for three days at 20 degrees Celsius. Move worms to a clean unseated NGM plate and incubate at 20 degrees Celsius for at least 30 minutes. Turn on the inverted microscope, the dissection microscope and pico injector.
Turn the selector knob of the pico injector to p clear and check that the current measurement is in PSI. This clear pressure reading is the input pressure and should be approximately zero PSI. Check the primary valve on the nitrogen tank regulator to make sure that the output is closed.
Open the main nitrogen tank valve, the regulator gauge nearest the nitrogen tank. Should now read the internal pressure of the tank. Slowly increase the out pressure by turning the primary regulator valve clockwise while monitoring the increasing pressure on the pico injector.
Slowly increase the pressure to 100 PSI when 100 PSI is reached. Switch the selector to p inject and set the injection pressure to 30 PSI. Using the p inject knob, switch the selector knob to P balance.
Place a 15 microliter droplet of mineral oil on clean donor and recipient plates. Cover a 2%agros injection pad in mineral oil. Load the pulled micropipet needle into the holder and align it under a dissection microscope mount one donor and one recipient worm near each other on the aros pad.
Position worms using low magnification and bring the needle into the mineral oil near the donor. Switch to high power and position the needle near a pseudo cavity. Increase the balance pressure to approximately 10 PSI.
A mineral oil plug will be visible at the tip of the needle. Push the worm into the needle to position the needle tip in the pseudo cavity by moving the stage. Note a jump in the position of the mineral oil in the tip of the needle.
Reduce the balance pressure to allow capillary action to fill the needle after collecting fluid. Slide the worm away from the needle. Once the needle is positioned in the recipient worm, use the injection function to inject the pseudo fluid collected from the donor.
Remove the needle from the recipient worm by sliding the microscope stage in the opposite direction as used to enter the recipient with the needle out of the worm. Raise the needle away from the injection pad. Remove the injection pad and place a droplet of M nine on the worms to recover.
Place a 10 microliter droplet of M nine on an assay plate. Pick the recipient worm into the M nine on the assay plate to assay for RNAI phenotypic Transfer. Allow the recovered worms to grow for 24 hours at 20 degrees Celsius.
Remove recipient adults leaving embryos and hatched progeny on the plate, incubate plate for an additional 24 hours at 20 degrees Celsius. Finally, score progeny as not hatched hatched, but being phenotypically a mutant or is wild type larvae shown. Here are results for experimental transfer of extracellular fluid from clear.
One E 1745 worms grown on bacteria expressing DSR targeting PAL one, the progeny of the donor animals died and or exhibited posterior patterning defects. After transferring extracellular fluid from these animals to the pseudo of RNAi naive wild type worms, a portion of the subsequent progeny of the recipient animal then displayed the expected PAL one mutant phenotypes. This is in contrast to the progeny of recipient animals who received extracellular fluid from donor animals grown on either standard bacterial food or control.
RNAi vector bacteria who displayed only background levels of lethality while progeny of recipients of extracellular fluid from donor animals undergoing RNAi show a significant increase in the frequency of DS RNA induced phenotypes. The penance is not as strong as in the donor animals progeny, where nearly 100%of progeny die as unhatched embryos in only rare, severely deformed animals hatch. After watching this video, you should have a good understanding on how to isolate extracellular fluid and perform inner animal transfers to answer questions about pseudos fluid makeup and function.
