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.
The goal towards my research is to identify new genes involved in reproduction. To do this I will be using the model organism Caenorhabitis elegans. They are considered a model organism because of their short life cycle, transparency (which allows for visual observations), ability to be easily manipulated, large amount of progeny production and the similarity between their genes to those of other organisms. The hopeful goal for this research would be to find genes in C. elegans, which affect reproduction, and to one day apply those findings and compare them to common genes in humans. These findings could potentially make headway in aiding infertility.
As of currently, I am continuing the research I began in the summer of 2015. This experiment involves the use of RNA interference (RNAi) with two C. elegan strains, spe-8 and rrf-3. The bacteria (E. coli), which the C. elegans eats, will be treated with a specific RNAi. Then over the course of three days, the visual effects that the RNAi is having on the targeted gene will be observed. What I am essentially looking for is an increase or decrease in the amount of progeny and eggs, both fertilized and unfertilized, produced after exposure to these RNAi treatments. Later on during my research I will learn how to photograph the worms to capture the physical effect on the strains.
So far in my research I have been able to not only expand on my topic but collect some data from experiments that I have set up. I began by furthering my research into what exactly makes a spe-8 C. elegan different from other strains. I was able to find that this mutant can trans-activate and form functional sperm during mating with males. In other words, spe-8 worms lack the ability to produce functional sperm to self fertilize so they are crossed with other males. To simplify even further, you can say these worms are “female” despite the fact they are not. C. elegans can only either be a hermaphrodite or male. However, there is a <0.2% chance that a male will be produced. This statement was key in understanding my results.
Again in my experiment I am using two different strains, spe-8 and rrf3. The males which I use are from the rrf3 strain while the hermaphrodites are from the spe-8 strain. Since my last post I have been able to successful set up a schedule in which I can record my data most accurately. So on a Wednesday at around 8 pm I will inoculate my L440 and M05D6.2 bacteria and Thursday morning at 8 am I will remove the bacteria from the 37-degree incubator. Optimal growth for these treated bacteria are 12 hours, shaking in a 37-degree incubator. Thursday night I spot my RNAi plates with a single 100 microliter spot of bacteria and let them dry at room temperature over night. 6 plates for L440 and another 6 for M05D6.2. On Friday I set up my crosses, which are the same as I had stated in my previous post. Then on Monday I pick off all of the worms that I placed on the plate, except for the new progeny (if there is progeny). Then on Tuesday I count my plates.
A constant result that I am finding is that I will have no progeny or eggs produced on the L440 plates with a single spe-8 worm. Then on M05D6.2 plates I find that there is a large number of unfertilized eggs, ~100-200. This result is consistent with a finding made in the “Characterisation of Caenorhabditis elegans sperm transcriptome and proteome. BMC Genomics.” paper. It states, “By RNAi of hundreds of sperm enriched genes, we identified a few genes, whose knockdown leads to production of unfertilized oocytes and significantly reduced broad size.” On the L440 and M05D6.2 plates there is a decent amount of progeny produced. However, it seems that I have a larger amount of progeny on the L440 plates then the M05D6.2 showing that M05D6.2 does effect fertility. If not previously stated, I am using L440 as a negative control. For a later experiment I will begin mimicking single nucleotide variants of the TCP11 gene, which again is the ortholog to M05D6.2.
Since the first post, I have chosen a specific gene and set up an experiment using RNAi. The gene that I am working with is M05D6.2; another name for this gene is CELE_M05D6.2 and is used in protein coding. There have also been human orthologs to this gene which have been found: TCP11L2, TCP11, TCP11L1 and TCP11X2. To compare my results using this gene I used L440 as a positive control. Also, I used two different strains of c. elegans during my experiment, rrf-3 and spe-8. It has been shown that rrf-3 has a homolog that is a hypersensitive RNA- directed polymerase, which inhibits somatic RNAi. Spe-8 is required for hermaphrodite spermatogenesis.
For this experiment what I had done was set up four different crosses between the rrf-3 and spe-8 worms using the RNAi treated bacteria, either being M05D6.2 or L440. The crosses, which I tried to do, were as follows. A spe-8 with an rrf-3 male, on a M05D6.2 plate and L440 plate, then a spe-8 on a M05D6.2 and L440 plate. In total I was to have 12 RNAi treated plates. On the plates with the cross between rrf-3 and spe-8, I was to set up three plates with one male rrf-3 worms with one spe-8 worm on both the M05D6.2 and L440 plates, 6 plates in total. For the solely spe-8 plates, I was to set up three plates with just one worm on each for both the M05D6.2 and L440 plate, six plates in total. However, when I went to set up the experiment I was successfully able to set up the spe-8 plates, but was only able to find 4 males in total for the crosses. So instead of doing three of each cross I did the three crosses on the M05D6.2 plates and one cross on the L440 plate.
The results of my experiment were as follows. On the M05D6.2 cross plate there were only 12 eggs on the plates. On another M05D6.2 cross plate there was only one worm on it. On one of the M05D6.2 plates with a single spe-8 worm there was only one worm. Then on one L440 plate, with a single spe-8 worm, there were twenty-five worms on it. The rest of the eight plates had neither progeny nor eggs on them. I must run this experiment a few more times to get conclusive results because the results were a little strange. However, the next time I perform this experiment I will cross the rrf-3 worms to get a larger production of males to use, while also watching for contamination. Also, instead of doing a cross with one spe-8 and one male rrf-3 worm I will do a cross between one spe-8 and four rrf-3 worms.