This video demonstrates a procedure for identifying genes that regulate, see elgan growth and body size. Using RNA interference or RNA eye to inactivate candidate genes first plates containing bacteria that expressed double stranded RNA, that target the genes of interest are prepared. Fourth larval stage hermaphrodites are transferred to the RNAi plates.
Once they reach the young adult stage, they're transferred to new RNAI plates to lay eggs. After several hours, they're removed leaving synchronized populations of embryos. At the desired stage of development, the progeny collected and mounted on slides.
Finally, microscopic images of the worms are acquired. Analysis of the image demonstrates changes in body size caused by gene inactivation. Though this method can be used to identify regulators of the TGF beta signaling pathway, it can also be applied to other pathways that regulate post embryonic development in sea elegance, such as the insulin signaling pathway.
Begin this procedure by cloning the target gene sequences into vector L 4 4 4 0 A commonly used worm RNAi plasmid transform the recombinant plasmid into the bacterial strain. HT one 15 DE three culture. The transformed bacteria on LB agar plates with 25 micrograms per milliliter of carbonic and 12.5 micrograms per milliliter Tetracycline, then pick a single clone for future use.
Meanwhile, transform the empty vector L 4 4 4 0 into the bacterial strain. HT one 15. To use as a control for the experiments culture, the transformed bacteria in one milliliter of LB broth with 100 micrograms per milliliter of ampicillin overnight at 37 degrees Celsius.
Tetracycline is not added because it decreases RNAI efficiency. Add another five milliliters of LB broth with 100 micrograms per milliliter of ampicillin to the overnight Culture then incubate for another four to six hours at 37 degrees Celsius. Once the culture is ready, seed 0.5 milliliters of experimental or control bacteria onto the RNAI worm plates labeled with the appropriate clone name.
At least two plates should be prepared for each condition. Incubate the plates overnight at 37 degrees Celsius. The next day.
A lawn of bacteria should be present that express RNAi with or without the target gene sequence. The animals will use the bacteria as a food source. Once the RNAI feeding plates are ready, flames sterilize the tip of a platinum wire.
Then use the platinum wire to transfer six to ten fourth larval stage or L four C elgan hermaphrodites to each RNAi plate. Let the hermaphrodites grow at 20 degrees Celsius overnight. The next day, the animals will be young adults.
Transfer them to a new appropriately labeled and prepared RNAI plate. Incubate the plates at 20 degrees Celsius for four to six hours to allow the adults to lay eggs. Following the incubation, remove all of the adults from the plate to synchronize the progeny.
Once the adults are removed, start to count time. This is time zero. Incubate the plates at 20 degrees Celsius to let the animals grow to the developmental stage of interest.
Here, young adults will be chosen for phenotypic analysis. For knockdowns that develop at a normal rate, 72 hour incubation is required. Keep in mind that various genes affect animal development differently, so incubation times may need to be adjusted.
If the RNAi causes animals to grow at a different rate, young adults can be identified as those no more than 24 hours past L four with completed VUL development and two to six embryos in the uterus as shown here. To identify other developmental stages, the investigator should use gonadal and VUL development as a guide. Once the animals have grown to the appropriate stage, prepare to score the body size phenotypes of the RNAi treated worms.
Next place two layers of colored label tape on a glass, one millimeter thick specimen slide as shown here. Make two of these glass slides. Then place a new glass slide between the two slides with the tape and apply a drop of 2%Agros melted in water.
To the center of the new glass slide, press a second glass slide on top to make a thin agros pad. Once the agros has solidified, remove the top glass. Slide the slide with the agros pad is ready to load worms.
Label the slide with the clone name. Add 10 microliters of 25 millimolar sodium azide to the aros pad. Then using a platinum wire as before, transfer 30 to 40 animals into the sodium azi solution.
To immobilize them, put a cover slip on the top. Do this for both recombinant and empty L 4 4 4 0 carrying RNAi treated animals. Next, place the micrometer slide under the dissecting microscope with a 2.5 x objective lens.
Using Q Capture or similar software, capture an image of the micrometer ruler and save it. Then place the worms under the scope and capture images. To measure the body length first, open the image of the micrometer ruler in Image Pro software.
Then click on measure and choose calibration. Then choose spatial Calibration wizard with calibrate the active image selected. Click on next.
Input the name for the calibration and ensure that the spatial reference units are set to microns. Then check the Create a reference calibration button and click Next. Click on draw reference line.
A reference scale bar will appear. Reposition the scale bar to match the ends of the micrometer. Then indicate that the reference indicates 1000 units.
Click okay. Next and finish. Once the software is calibrated, open a worm image.
Then under the measure menu, select calibration, and then click on select spatial. In the pull down menu, select the file name created for the micrometer calibration. Then under the measure menu, select measurements in the window that opens.
Select the free draw tool and using the computer mouse trace a line through the center of the animal body from head to tail. The length will be reported in the window. Repeat this process for all animals in the field.
Export the length measurements to a Microsoft Excel file or other suitable statistical software. Analyze the data by calculating the mean and standard deviation for each sample. Then compare the results for each group using a student's T-test.
The transforming growth factor beta or TGF beta SUPERFAMILY protein DBL one is required for activation of the TGF beta pathway. In response to signal activation, intracellular smad signal transducers, translocate to the nucleus and initiate gene transcription. See elegance in which the TGF beta pathway is disrupted have a smaller body size, including a shorter body length than wild type animals.
Thus screens for sea elegance. Body size mutants are capable of identifying TGF beta signaling components and modifiers to test the effectiveness of RNAi by feeding for the identification of body size mutants. RNAi was used to knock down DBL one pathway components SMA three at SMA six in four different strains of sea elegance.
The C elegance strains tested included two that are RN AI hypersensitive A one, Lin 15 B, and RF three, as well as the standard wild type strain N two and the LIN 36 strain, whose RNAi sensitivity is similar to N two as shown here. When SMA three or SMA six expression was disrupted, the animals displayed a significantly shorter body size than control animals except SMA six RNAi in Lin 36. Background in RF three.
Background body lengths of young adults after RNAi treatment were 84 to 95%of vector alone. In A one Lin 15 B background body lengths of young adults after RNAi were 68 to 86%of the control animals. However, the A one lin 15 B strain produces view progeny.
This renders them unsuitable for large scale screening. Therefore, the use of the RF three strain is suggested for these techniques. After watching this video, you should have a good understanding of how to identify genes that regulate growth and body size using RNI interference technique.
