In the previous blog post, I walked you through the manner in which I would set up my next experiment. In that experiment, I exposed five N2 L4 hermaphrodites to three treatments; L440 (negative control), npp- 19 (positive control), and M05D6.2 (gene of interest), using RNAi. From that experiment, what I was able to conclude was that the M05D6.2 treatment did not have a significant impact on N2 hermaphrodites in regards to fertility. This finding helps support my claim that the M05D6.2 gene does not impact hermaphrodite fertility but does impact male fertility. The progeny counts for the M05D6.2 and L440 treatments were very similar. To show the significance of this data I will use the program R to perform statistical analysis on the collected data.
Along with collecting this data, I performed microscopy on male C. elegans, using the strain AJ740. Prior to viewing the worms, they were exposed to an RNAi treatment using the gene M05D6.2. This strain was used due to the fact that they have a GFP tag on their tubulin, allowing them to fluoresce when being viewed under a fluorescent microscope. What I was looking for within these males was a decrease in sperm production. From the images that I captured I did see that this strain was able to produce sperm after being exposed to the RNAi treatment, but to properly say if there was a decrease or not in sperm production I will have to repeat this experiment.
In the following weeks, I plan to set up a number of different experiments. One of those experiments is to cross five N2 C. elegans males with spe-8 hermaphrodites. Spe-8 worms cannot properly produce their own sperm, so if a spe-8 animal self-fertilizes it produces a “dumpy” worm as opposed to if it were to mate with a N2 male, producing a normal sized worm. So, if there is a larger count of dumpy worms after the cross then we can say that silencing the M05D6.2 gene causes an impact on male fertility. Because a higher number of dumpy worms means that they hermaphrodite was self-fertilizing as opposed to crossing with the N2 males. Another experiment I want to do is creating another replicate of my initial experiment where I crossed five rrf-3 males (RNAi sensitive males) to a spe-8 hermaphrodite. After setting up these experiments I will pick off the males that were used and DAPI stain them. By DAPI staining the males it will allow them to be viewed under a fluorescent microscope.
Since the last blog post, I have made progress in my scientific research. Before taking on new experiments, my research mentor and I decided to sort through old data I had previously collected in an aim to figure out if any experiments, I had conducted in the past, needed to be repeated. This was done to make sure that I had enough replicates of my experimental data to produce more statistically significant results. After reviewing my data, I chose to replicate an RNAi experiment using my model organism of choice N2, C.elegans hermaphrodites. Again, N2 C. elegans are a wild-type strain. What this experiment involved was using a positive control (npp-19), a negative control (L440), and my gene of interest (M05D6.2) to view the amount of progeny that was produced after five days of experimentation.
To set up this experiment, the first thing I did was grow my bacterial cultures, which contained siRNA, that would be used to silence a targeted gene, on a Monday evening. The bacteria were then incubated for 16 hours. After 16 hours a 100 microliter spot of each bacterium was placed on a RNAi plate and incubated at room temperature for 24 hours. On Wednesday morning I set up my experiment, placing 5 L4 N2 C.elegans hermaphrodites on each plate and incubating them at 22 degrees Celsius overnight. On Thursday I picked off the matured C. elegans hermaphrodites, leaving on the plates the eggs which had been laid. Then on Friday I counted the amount of hatched and unhatched eggs on each plate. The results of this experiment showed that silencing the M05D6.2 gene in N2 hermaphrodite C.elegans had no effect in progeny count. This is exactly the result we wanted to see. Showing that this gene does not play a role in hermaphrodite fertility helps solidify our previous conclusion that the M05D6.2 gene plays a role in male fertility.
Along with collecting the data from this experiment, I have also been performing statistical analysis using a program called R. The next step in my research will be to learn how to use a technique known as microscopy to view internally the impact of the RNAi treatments on male C. elegans as well as hermaphrodites. I will also be setting up a new cross between two different C. elegans strains to see how the fertility of these two strains are effected after being exposed to the RNAi treatment.
The research that I am conducting in the academic year of, 2016-2017 in the UGR research program is a continuation of the research I performed during the academic year of 2015-2016. That research involved looking into the potential function of the M05D6.2 gene and its connection to the TCP11 gene in humans. To perform this research, we used the model organism of C. elegans. We compared the M05D6.2 gene to the TCP11 gene, which is the human ortholog of M05D6.2, to see its impact on fertility. At the end of the year, the preliminary data that we collected allowed us to conclude that we believe the M05D6.2 gene plays a role in male infertility. The approach we used in making this conclusion was by using RNAi, more commonly known and RNA interference. What we were looking for was a decrease in progeny count in the C. elegans that were exposed to the M05D6.2 treatment.
Currently, we have commenced our research by first looking at the effect of RNAi on N2 hermaphrodites. The N2 strain, of C. elegans, is considered to be the wild-type of this organism. We have previously used RNAi on other C. elegans hermaphrodites, from different strains, but we have not yet seen that effect of RNAi on wild-type hermaphrodites. In the hermaphrodites, from the different strains, we saw no decrease in the amount of progeny that was produced. This means that in these hermaphrodites the RNAi had no effect on fertility. Another thing that we are interested in is looking at how the internal structure of the organism, more primarily the sperm, is being effected by the RNAi. To see this change we want to image the worms. We can do this because C. elegans are transparent and their transparency makes it easy to see their internal structure. In humans, it has been found that the TCP11 gene impacts the way in which a sperms tail coils, thus if this gene is mutated it makes the sperm infertile, because it can no longer function properly. The two ideas of interest, I have talked about here, are only two of many that my team wants to investigate.