The goal of this procedure is to identify post embryonic regulators of protein expression and localization in C elegance using an RNAi based genomic screen and fluorescently tagged proteins. Once the appropriate screening strain is constructed, a an RNAi library is chosen. The first step of the procedure is to package the library clones into bacteria.
The bacteria are then selected, grown up, and fed to staged c elegance nematodes. After allowing the nematodes to grow for 72 hours, they are observed for alterations in the phenotype of interest. Ultimately, changes in the expression or subcellular localization of attacked protein are visualized with fluorescence microscopy.
This method can identify genes required for the proper subcellular localization of a fluorescently tagged protein. However, this protocol can be modified to identify genes affecting other post embryonic phenotypes of interest. The main advantage of this technique over existing published protocols is that we have optimized the consistency and level of the RNAi response in animals by inducing production of the double stranded RNA in bacteria before plating.
Demonstrating the procedure Will be Kathy fuss, a research associate from my laboratory Within two months of beginning the experiment, prepare the clone library first streak bacteria from selected feeding library clones, as well as positive and negative controls on LB carbonic tetracycline plates. For a positive control, use a clone containing a gene that produces a dose dependent post embryonic phenotype, such as BLE four. For a negative control, use an empty vector or a clone containing a predicted pseudogene with no observed phenotypes.
Incubate the plates overnight for each clone inoculate two milliliters of LB medium containing 50 micrograms per milliliter. Carbonic acid with a single colony from the streak plates, incubate the cultures overnight with agitation the next morning. The solutions should be cloudy induced production of double stranded RNA by adding IPTG to the cultures and incubate them for an additional four to five hours under the same conditions.
Adding IPTG ensures a more consistent and robust RNAI induced knockdown of gene product than relying solely on the IPTG and the auger of the NGM plate After the IPTG incubation Spot 30 microliters of culture from each clone into each well of the prepared 24 well NGM RNAi plates. One clone per well. Each plate should have two wells devoted to the controls.
Carefully ma the location of each library. Clone in the 24 well plates now place the plates uncovered in a sterile flow hood to dry for about 20 minutes. Do not allow the edges of the agar to pull away from the plate.
Once dried at staged nematodes to the plates, instructions on nematode preparation are given. In the next section, Start with a staged population of first larval stage or L one nematodes. Beginning with L one larvae bypasses potential embryonic lethal phenotypes.
Having a staged population of animals to screen also increases one's confidence that any variations seen are due to the RNAi and are not simply normal variations that occur in different stages. However, if the RNAi causes a developmental delay, this should be noted by the screener Bleach. A mixed stage population of the screening strain.
Only eggshell protected embryos will survive this step stages animals to within 12 hours if conducted at 20 degrees Celsius or to within 18 hours. If conducted at 16 degrees Celsius on the day that bacterial cultures are inoculated. Select three 100 millimeter plates of screening strain animals that are healthy and contain both gravity, adults and embryos.
Wash all the animals in sterile M nine media and transfer the wash to a sterile 15 milliliter tube with a screw cap. If the wash is greater than 3.5 milliliters, spin on the animals and remove all but 3.5 milliliters of liquid. If less than 3.5 milliliters.
Increase the volume to 3.5 milliliters with water or M nine to free the embryos from the adults at 1.5 milliliters of a mixture of sodium hydroxide and bleach to each wash. Allow the animals to incubate for no more than 10 minutes in this solution. After starting a timer, vortex each tube for 10 seconds and repeat the vortexing every two minutes.
After each vortexing, observe the solution under a dissecting scope for animal carcasses. Within six to eight minutes, the adults will be fragmented and the embryos should be released. Remove the embryos from the bleach solution by pelleting them and removing as much of the supernatant as possible without disturbing the pellet.
Then resuspend the pellet in a volume of 10 milliliters by adding sterile M nine and using agitation. Repeat this wash process at least once more until the smell of bleach is completely undetectable. To more tightly stage the animals arrest them in their L one stage by hatching embryos in M nine medium without food.
This is done by transferring the embryos in M nine to a small erlenmeyer flask. Shake the flask overnight at the proper temperature for the strain and experiment. In this time, the animals will all hatch and arrest in the L one diapause the next day.
Resume the larvas growth from a set time point by transferring them to the prepared bacteria expressing double stranded RN.This is the same day that the 24 well plates have been spotted with bacteria. Start by transferring the L one animals to a 15 milliliter tube and spinning them down. Then concentrate the animals into one half to one milliliter of M nine and place five microliters of concentrated nematodes on a plate.
Count the number of animals on the plate and adjust the concentrated nematode solution. TE 30 worms for every three to 10 microliters. Finally, spot approximately 30 L one stage animals onto the bacteria in each well of the prepared.
24 well plate more than 30 animals per well may result in starvation After the spots have dried, which will take a few minutes, secure the lid with the rubber band incubate the animals inverted at the required temperature and duration to score the animals at the desired stage. Once the nematodes are ready to screen, confirm that the positive and negative controls produce the expected phenotypes. Then observe the experimental animals for developmental abnormalities.
Next, using a dissecting microscope mount eight to 12 animals from each well onto 4%Agri PET slides with a five microliter drop of anesthetic. After applying the appropriate cover slip, make observations of at least five animals using compound microscopy. Keep an organized database of the gene, number of animals observed and phenotypic results.
For each RNAI experiment, verify any R RNAi produced phenotype by another secondary phenotype if possible, and by repeating the experiment, repeated experiments can use bacteria from the same streak. Older streaks may result in poor growth in liquid overnight cultures, and should be re streaked first. Some bacterial cultures won't grow well because of the gene product expressed in their plasmid.
In this case, simply concentrate the bacteria before plating. For an ongoing screen, it is important to keep plenty of embryos always ready for staging. The screening strain should always be fed, not starved, and free of contaminants that may affect the phenotype of interest.
We established a maintenance schedule for feeding animals. Genes that affect DBL one localization were identified by RNAi using a strain design for this screen. Normal expression of GFP tag dbl one includes ventral nerve cord cell bodies, and a row of puncti animals fed antisense RA to DBL.
One mRNA showed severely attenuated expression of DBL one. These animals were also small like animals lacking functional DBL L one. The RNA I screen successfully identified many other genes whose products affect DBL one localization, thus expanding our understanding of how TGF beta is subcellular trafficked and providing a launch point for many more investigations.
Using this method, a single person can reasonably stage and observe two to four sets of experiments every week. For instance, animals staged on Monday and Tuesday can be observed on Thursday and Friday respectively, and animals can again be staged on Friday and Saturday for Monday and Tuesday. Observation While following this procedure throughout your screen.
It's important to keep plenty of embryos ready for staging, so maintain the screening strain regularly. Also, it's important to take detailed notes on all observations.
