His diligent guidance and abundant patience in helping me understand complex processes and techniques made it possible for me to complete this thesis. I would like to thank Jamie Wood for her guidance and mentorship towards the end of her time at Ole Miss. And, of course, I would like to thank my parents for supporting me throughout this process and helping me get here in the first place.
The development of a complex nervous system requires the actions of complex genetic mechanisms that influence and maintain the differentiation of common nervous system progenitor cells into neurons and glial cells. In Drosophila melanogaster, the gene glial cells missing (gcm) encodes the transcription factor Gcm which, when active, causes early nervous system cells to preferentially differentiate into glial cells by. However, the Gcm protein also plays a role in the differentiation of both macrophages and tendon cells.
PBS phosphate-buffered salmon PCR polymerase chain reaction Pen/Strep penicillin/streptomycin PNS peripheral nervous system PNT PBS/NGS/Triton.
INTRODUCTION
In the Drosophila nervous system, one gene, lacking glial cells (gcm), is primarily responsible for determining neuroprogenitor cells to become glial cells. Repo and Pntp1 work together to regulate the expression of the loco gene, the encoded protein of which is necessary for glial cell morphogenesis (Lee and Jones, 2005; Jones, 2004). Although repo is exclusively expressed in all lateral glial cells (Jones, 2004), gcm also plays a role in tendon cell development.
The pAbAi vector carries the reporter gene Aureobasidin A resistance, which confers the ability to grow in the presence of the Aureobasidin A antibiotic (Nipper, 2014). When a fusion protein binds to the “bait” sequence, it causes expression of the AbAi reporter gene, conferring the ability to grow on selective media containing Aureobasidin A (Nipper, 2014). A yeast expression vector carrying the gcm gene was introduced by transforming it into the Y1HGold/AbAi yeast reporter strain, creating another dimension in the screen (Nipper, 2014).
One of these screens produced yeast colonies in the presence of Aureobasidin A containing both gcm and groucho cDNA. Although the groucho fusion protein induced resistance on its own, suggesting that it may interact with endogenous factors, resistance was significantly increased when both gcm and groucho were present, suggesting a synergistic interaction between gcm and groucho in yeast cells (Nipper, 2014). Gro, the Drosophila member of the Groucho family, is present almost ubiquitously within cells and is required for nearly every aspect of embryonic development, including sex determination, neurogenesis, segmentation, dorsoventral patterning, terminal patterning, eye development, and wing development (Song et al., 2004).
Gro, like other members of the Groucho family, is a repressor, and most evidence to date suggests that Gro mediates long-range repression (Song et al., 2004). Dicer has four domains: an amino-terminal helicase domain, two RNase III motifs, a dsRNA-binding domain, and a PAZ domain ( Agrawal et al., 2003 ). This complex is believed to be activated in the presence of ATP, exposing the antisense portions of the siRNAs.
If possible, cDNA corresponding to the groucho sequence should be used for PCR amplification (Worby et al., 2001). The loss of the protein can then be measured directly using a Western blot analysis, which requires an antibody specific for the protein, or by staining the cells to observe protein loss (Worby et al., 2001).
MATERIALS AND METHODS
A 1 µL sample of the PCR product was then run on a 0.8% agarose gel to confirm that the reaction had yielded the product of the correct size. To precipitate the RNA transcript, the following steps were performed similar to PCR DNA precipitation, but with slight changes. 1 µL of the resuspended RNA transcript was then run on a 1% agarose gel, along with a 2 µL sample of the ready-to-use RiboRuler high range RNA grade provided in the kit and 1 The other μL was used with a 1 kb DNA ladder.
Next, a test annealing reaction was performed by combining 0.5 µL of RNA transcript and 1 µL of 10x annealing buffer (1 M potassium acetate, 300 mM HEPES–potassium hydroxide, 20 mM magnesium acetate, and DEPC-treated water to 100 mL). For regular maintenance of the cells, the flasks were removed from the incubator every 48 h and the cells were washed from the bottom of the flask by repeatedly pipetting the cell solution up and down while holding the flask at an angle of approximately 45 degrees. The supernatant of the old medium was removed, leaving approximately 2 ml of the old medium in the test tube.
An 8 mL volume of fresh complete S2 media was then added to bring the volume to 10 mL, and the cells were resuspended by pipetting. A 5 mL sample of this cell solution was then added to 10 mL of complete S2 medium in a T-75 flask and incubated at 25 ºC for another 48 h. After two transfers, the cell culture was split to form a backup culture in case one of the cultures became contaminated.
400 µL of the diluted cell sample was added to each well of a 12-well cell culture plate and incubated at 25 °C for 24 h. A 75 µL sample of cells from each well of the tissue culture dish was added to a complementary well on the slide, with wells 1-6 positive for RNAi and 7-12 negative for RNAi. While holding the slide flat, 2 mL of 1x PBS was pipetted onto the top of the slide and the excess gently poured off.
A 15 µL sample of the primary antibody was added to each well, and the cells were incubated in the humidity chamber for one hour. A 15 µL sample of the secondary antibody (CY3 goat anti-mouse), diluted 1:1000 in PNT, was added to each of the wells on the slide, and it was also incubated for 1 hour in the humidity chamber at room temperature.
RESULTS
The second last RNA band (black star) seen with the DNA ladder (Fig. 4B) is approximately in the same position relative to the ladder as the double-stranded DNA (Fig. 4A). Because the remaining DNA should have been degraded by the previously described DNase reaction, this suggests that this band is likely to be the desired dsRNA complementary to the gro target sequence. Part C shows the products of the same transcription reaction next to a single-stranded RNA ladder.
The annealing reaction, when performed on a 1% agarose gel (Fig. 5), revealed that all of the single-stranded RNA did not anneal. However, the relative brightness of the upper and lower bands changed compared to their brightness before the annealing reaction (Fig. 5). The lower band (ssRNA) was brighter than the upper band (dsRNA) before the annealing reaction (Fig. 5, far right lane), but the upper band appears brighter than the lower band after the annealing reaction (Fig. 5, middle). Lane).
This suggests that although the annealing reaction was not fully effective, some of the previously ssRNA did anneal to form dsRNA, which combined with the already dsRNA that likely self-annealed during the transcription reaction. After performing RNAi on the Drosophila S2 cells, they were examined under the fluorescent scope. This shows the results of running on a 1% agarose gel the products of the annealing reaction performed on the RNA obtained by transcription.
The products of the annealing reaction (very right lane) are compared with the products of the transcription reaction prior to annealing (middle lane). The products of the annealing reaction performed on the gel were more dilute than the transcript products, explaining the reduced brightness. Thus, the results of the experiment are difficult to determine and are considered inconclusive because it appears that the experiment was not actually performed on Drosophila S2 cells.
DISCUSSION
This suggests that the band contains ssRNA from the transcription reaction because it represents the target size on the single-strand scale. This suggests that the band contains a 744 bp long segment of dsRNA, which would have appeared longer compared to a single-stranded ladder because double-stranded molecules are larger and take longer to moved through the agarose gel. The other visible bands above these two aforementioned bands may have come from some of the ssRNA forming secondary structures within itself, causing it to become bulky and move slowly through the gel.
When attempting to anneal the remaining ssRNA so that the entire sample would be double-stranded, the reaction did not appear to be completely efficient. Because the S2 cells are derived from a macrophage-like lineage and exhibit a similar phagocytic nature (Schneider, 1972), they should have taken up the dsRNA molecules during soaking, which could then take part in the RISC complex. After staining the cells to examine the results of the RNA interference, viewing them under the fluorescent scope gave inconclusive results.
Some previously stained S2 cells were examined to see what the results should look like, and this. It is likely that the S2 cells became infected at some point and were overtaken by cells of a different type, which is supported by the fact that the cells appeared to multiply faster than usual and were a slightly different shade of color has. It is not likely that the cells underwent a transformation due to epigenetics, as these differences were also seen in the RNAi samples.
It would also be better to try to do the actual experiment after maintaining the S2 cells for a maximum of three weeks to reduce the risk of. If the RNAi experiment is performed with positive results, the ultimate goal would be to examine the effects of groucho knockdown on repo expression by staining for the presence of Repo in the cells. Culture of Drosophila S2 cells and their use for RNAi-mediated loss-of-function studies and immunofluorescence microscopy.
