Blog #3: Determining the function of F10C2.4 in reproduction

Since my last blog post, I have made promising progress with my research project. My main goal of this semester was to focus on microscopy. Now that I have established that my gene of interest, F10C2.4, clearly shows some kind of reproduction problem when not present using RNA interference, I wanted to utilize the unique feature of my model organism, Caenorhabditis elegans, to determine what is going wrong in the organisms without this gene. One of the drawbacks from using technology such as a microscope hooked up to a computer is that sometimes things can go wrong; however, I feel that I have learned so much from the mistakes, which has helped me get a better understanding of the research I do.
Caenorhabditis elegans have many benefits for genetic manipulation and research. One of the most beneficial features is that it is transparent. This is great for microscopy because it makes it easier for us to see what is different with the worm’s reproductive system when comparing it to the normal, not treated worm. For the experiments I perform for the microscopy element, we repeat the RNAi interference experiments with strains with fluorescent markers. GFPs are green fluorescent proteins that can stain a particular part of a cell; like a cell wall and RFP are red fluorescent protein can stain the chromosomes within the nucleus of the cell. With the strain I am working with, AJ740, I can utilize the GFP and RFP to see what is happening to the shape and overall placement of the eggs within the affect mother worm treated through RNA interference along with what is going on with the chromosomes. I have several questions. General questions like: are the eggs going to the right place and are there too many or too little eggs in the mom’s body? In regards to the chromosomes, are they separating at the right time? Is there an issue that is causing the incorrect amount of chromosomes in the cell which is not allowing them to hatch once they are laid? Does the egg go through the correct amount of cell divisions before being laid? All these questions can be answered using microscopy thanks to the organism, C. elegans.
A challenge that I have been facing, along with other members in my research group, is in regards to getting the microscopy aspect to work. This has opened my eyes to the many components involved in a task like this where things can go all good for one week but the next week, multiple things can go wrong. That being said, it is important to troubleshoot what went wrong and come up with ways to fix the problem at hand. An example of this is when the GFP and RFP do not show up bright enough to identify anything going on in the C. elegans. This could be due to the organisms not growing properly due to a lack of food or temperature sensitivity. Whichever the case, it was important for us to work together as a team to come up with a way to avoid this issue for the next week. For the future, I hope I can take more images from microscopy to look further into what is going on internally to determine what the function is of my gene of interest, F10C2.4.

Blog Post #2- Determining the function of F10C2.4 in reproduction

Eight weeks ago I discussed my three goals for this year. First, analyzing the data I have found so far. Second, performing experiments using a strain related to my gene of interest from the Million Mutation Project. Third, perform microscopy to further my understanding of how reproduction works in the C. elegans. So far I have started to work towards each of my goals in small steps.
My first goal of analyzing the data I had collected so far along with a few repeated experiments from this semester. I has already collected similar data from about three trials from the academic year 2015-2016 however I wanted to confirm that the RNA interference was the same as I had established last semester. With the help of my research team, we were able to repeat the experiment three times with identical results. That being said, we can safely conclude that my gene of interest F10C2.4 plays a role in interrupting something in reproduction. With the RNA interference experiment we looked at two controls, the negative control [L440] and positive control [npp-19]. L440 is designed to show no issue in reproduction. As a result we expected to have a large percentages of the eggs on the plate to hatch into worms. Npp-19 is designed to have a problem in reproduction where there is a problem with the mom and that causes eggs to be laid but not hatch on the plate. With RNA interference with my gene F10C2.4 I got the results of npp-19 thus resulting the conclusion mentioned previously. By analyzing the data, it can make it easier for people to understand what it really means as a whole rather than individual trials. I have learned how to perform exploratory data analysis and now we are looking for the right program for statistical analysis. Since I haven’t taken statistics yet, it makes the process a little more difficult which is why we are at a standstill in regards to that.
My second goal was to perform experiments using strains from the Million Mutation Project. MMP is unique that it is a way to understand genetics in C. elegans. I have worked on going through the various MMP strains and targeted the ones that had mutations in my specific gene of interest, F10C2.4. As a result, I had to narrow down the fourteen different strains I had depending on which had a conserved sequence in the human ortholog for my gene of interest. As a result I choose three to five genes to order and just on Friday we have received them. Now we need to prep the C. elegans making sure they are well feed and healthy to have experiments performed with them. That will be one of the first things we do next semester.
My third goal is something I have been meaning to do for a while since it can give us a lot of information about what happens in the C. elegans’ body. One of the great advantages from using this organism is that it is transparent which lets us see in their bodies very easily. By using microscopy we can look that the embryos of the mom C. elegans to determine at what stage does the cell division may have stopped or have an issue. From that we can infer more about what F10C.24 is necessary for. However due to technological problems with the microscope we haven’t been able to work on it but hopefully by next semester we can start.
So far, I have learned so much from how research in genetics works. There is a lot of different methods and experiments to perform with the model organism of Caenorhabditis elegans which is a benefit for us, the researchers. Although there has been some rough patches with the technology I am hopefully that next semester we will be able to move further towards visually seeing what happens when F10C2.4 is not active in the genome.