My summer project is a continuation of my work on C. elegans nematodes from last year, it is regarding a gene involved in cell cycle regulation.
At the beginning of my research I had looked up literature about the ortholog of my gene of interest in humans and it pointed to a dysregulation in cell division and ultimately, “mitotic arrest”.
When I ran the first RNAi experiments in which I would essentially “delete” the production of this gene in C. elegans’ embryos, the nematodes would produce eggs but the eggs would not hatch.
I then imaged the eggs, this showed that the eggs were being fertilized and actively dividing but they were not hatching. I also imaged the effects on the embryos of C. elegans that contained a fluorescent histone marker (histones are located on the DNA of every cell in the embryo), this allowed us to see some differences in the DNA between the embryos with a deleted gene and the wild-type embryos.
This summer I am using three different strains of Green Fluorescent Protein-tagged nematodes that display fluorescence in different areas of the embryo that will allow me to identify any other differences in embryo development, I will use this information to be able to better identify the characteristics and the role of this gene in cell division of C. elegans embryo.
We will be using RNAi extensively, we will also use Green Fluorescent Protein-tagged C. elegans and fluorescent microscopy to be able to image the embryos.
Throughout this year of research we had a huge progress on our research, I am very happy with the results we have had so far. We have had a positive result on our protein for regulation of cell division. This is promising for further characterization of the protein itself since it is an ortholog to a human protein.
We started being able to do new assays that have also been able to give us positive results and a more in depth look into our research, I am very excited to present our results at the Spring Showcase and to continue doing research during the summer and next semester.
The opportunity provided by the Provost Undergraduate Research has been nothing short of amazing. I am very grateful.
I am so happy to be doing research this semester! I have found that I have more time to get things done than I had in the Fall. I have been trying very hard to start from where I had left off in December but I have been running through some technical issues along the way. I am hoping that by the end of this week I can start seeing results to be able to move forward to my next step. The challenges have been hard to fix. Right now, I am trying to define the timing the worms are taking to mature, since it has changed, and this step is essential for the experiments to work properly. I have learned that research takes a lot of patience, and sometimes things do not get done when you expect them to. But its all part of the learning process, and its important to keep a positive attitude and to not give up!
During the past several weeks we have gathered information on the protein of interest by reading multiple peer reviewed research articles on the human protein and its function, the goal with this is to be able to have enough background information on the protein to draw out a more specific hypothesis, as well as specifically tailor the following steps in our research.
We have conducted a few successful RNA interference experiments, and we are in the process of being able to capture images of the live animals in which the protein has been deleted. This will allow us to get a clear visual examination. Thus far, all of the RNA interference experiments have shown a large decrease in progeny, about 92% less than the non-treated C. elegans.
At the moment, the questions we have are regarding the point in development in which the embryos are failing to develop.
During the background research we also discovered that this protein is responsible for DNA damage response and is a protein that is found in the cell cycle checkpoints, so we would like to be able to pinpoint its role in Caenorhabditis elegans embryo development.
The ultimate goal is to be able to provide a highly homologous model for research of this protein in C. elegans. We hope that by providing this model, the ability of this protein to interrupt rapidly reproducing cells, such as C. elegans embryos may assist in the research for cancer treatment alternatives.
The title of my project is Identification and characterization of a spliceosome protein orthologous to Homo sapiens in Caenorhabditis elegans.
This particular protein in H. sapiens has been discovered to have an important role in cell division, as a checkpoint protein between division stages. This protein is responsible for proper alignment and distribution of chromosomes during mitosis. An appealing target for cancer treatment given that the depletion of this protein causes cells to stop division and die. Presently used cancer therapy is focused on targeting rapidly dividing cells because cancer is the product of cell division without regulation.
This project is interesting given that the gene I want to study in the nematode C. elegans is a gene that is also found in the human genome. As I looked closer into its sequence, it has resemblance not only in the sequence but in protein properties as well, but it has yet to be identified that it has the same function in C. elegans
Caenorhabditis elegans over the last years has increasingly become the live model system of choice. They can be one of two different sexes, male and hermaphrodite, which means there is less amount of genetic diversity since the hermaphrodites are able to fertilize their own eggs. It has a rapid reproduction rate of about 300 worms per adult. And it has a clear cuticle, which allows us to view inside the animal without having to kill it.
At this point the research on the human protein is currently carried out in mammalian cells in vivo, which are difficult to maintain and very challenging to reproduce. By using a live model like the nematode C. elegans, we have an easier, more manageable approach. The purpose for this research is to be able to provide a live model for the study of this protein by proving it functions the same in C. elegans as it does in humans.
To be able to identify that this protein has the same function in both humans and C. elegans first we need to establish that the protein regulates cell cycle similar to human cells. To study protein function we will use RNA interference, or RNAi. In RNAi, a sequence of RNA is introduced into the animal and this results in the animal losing function of that specific gene. RNAi experiments will allow us determine how the gene functions. I will be focusing on embryo development in the nematode and identifying which process in cell division this gene controls.