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.