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.
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.
Last year (2015-2016) was a very successful year in achieving one of my goals thanks to the Undergraduate Research Program. My project is titled “Determining the function of F10C2.4 in reproduction.” This is a genetics based research in which I use the model organism Caenorhabditis elegans to work with my gene of interest (GOI) F10C2.4. Last year using RNA interference (RNAi) I was able to determine that F10C2.4 plays a role in reproduction, specifically in regards to producing viable offspring. This means when I performed the RNAi experiments which knocks out or takes out my GOI to allow me to determine what happens when it is not there. As a result I saw plates with very few worms and a large amount of unhatched eggs. This meant that the mom was able to lay the eggs but something went wrong so they cannot hatch and become progeny. F10C2.4 is a gene that we do not know its exact function. It is my goal to be able to determine its function so that it can eventually be named. F10C2.4 also has a human version of the gene. By determining the function in C. elegans then we can relate that to humans to understand its role in humans as well.
This year I plan on repeating the RNAi experiment one or two times to get a larger amount of data that can be further analyzed. There will be three focuses for this year. The first is to be able to work on analyzing the data and results I receive so that I can know the statistical significance. The second is to perform experiments using strains related to my GOI from the Million Mutation Project (MMP). Hopefully by looking at other mutations other than knock outs from RNAi I can get a better understanding of other functions of my gene. My third focus would be to look at the mother C. elegans under a microscope to determine if there are any physical changes that go on in the mom. The benefit of the C. elegans is that they are transparent which makes it easy for us to look at their internal body, especially their embryos [or eggs] to see if there is some kind to separation problem within the chromosomes which causes the defect. I can eventually perform RNAi experiments with Green Fluorescent Proteins (GFPs) that will make certain things like the outside of the embryo light up under the microscope which allows for easy tracking.
I am looking forward to working with the other members of my research group along with my adviser Dr. Matthew Marcello in continuing this project. Just from last year I have learned so much out of the classroom and with these new focuses I hope to learn even more to help me become a better scientist and researcher.
Over the past year I have been developing and improving my research methods in order to get a better understanding of each steps involved in my project. So far we have moved from understanding that F10C2.4 is involved in reproduction to gathering data to support that F10C2.4 is not necessary in ovulation however is necessary to produce viable offspring. From this the results have shown that there is a dramatic decrease of progeny when F10C2.4 is not present. In preparing for presenting my research at the Society of Fellows I had to dig a lot more into my gene to see what it does. From that I have learned that it is a DNA Polymerase which may be necessary for specifically replication or something else. F10C2.4 is a gene that encodes for a protein which makes up to delta subunit of DNA Polymerase. From the schematic online, that looks like a small part of the overall Polymerase. It makes me think how such a small change can impact the viability of an organism.
I have learned so far that scientific research takes a lot of steps and preparation along with online research to understand what is updating within my field. However it has been a growing positive experience in which I hope to continue to develop my project.
Compared to last time, we have developed our methods of RNAi and developed a process which works for the way I want to express my experiment. Previously, I was having a hard time having consistent results but by this time around we have found a way to repeat the experiment and have consistent results. Along with this, I have learned how to make the bacteria to go on the plates, how to spot them and look at the worms under a microscope. With a trend of a large percentage of unhatched embryos with my gene of interest, we know that it must have some type of role in development. Under the microscope we looked at the adult mother worms from the RNAi plates, both the control (L440) and gene (F10C2.4) to look at physical differences. In order to do so, I used a different strain of worms. Instead of the wild type N2, I used a strain that had a GFP (green fluorescent protein) that showed the embryos and eggs in the mothers body which made it easier to see under the microscope. While looking at both types of worms, we realized that there was still something funky looking in the controls even though they showed normal progeny development. That made it difficult to compare what we saw to determine what is normal and what isn’t. This makes me realize that it is hard to have a “perfect” model to compare to which organisms are able to have mutations that may not effect the worm’s phenotype. As a result, I need to do more experiments and look at the worms under the microscope to see the physical differences. From that I can pin point the exact way I want to approach determining how this gene works.
The focus of my research is to determine the function of gene F10C2.4 in regards to Reproduction in C. elegans. C. elegans are microscopic worms that are model organisms since they have their whole genome sequenced, reproduces quickly, easy to maintain along with can easily be manipulated and observed due to its transparency. C. elegans have about 40% the same genes as humans, therefore we hope to compare our results to how reproduction occurs in humans. This project will help me practice the skills in learn in Genetics lab on a gene of my own thus furthering my understanding of the methods learned in class. The major method we have used is RNAi treatment which knocks out the gene of interest. So far, our results have not been consistent therefore we are tweaking factors in the worms and environment to see what gives us the best result. Overall, with the research papers I have read so far, we know very little about this gene. Hopefully, during this year I will be able to gather some more information about F10C2.4 in order to determine why we have this gene, how does it effect reproduction and whether or not my results transferable to human cells.