In the past few weeks of the internship, I have continued to learn and improve on new lab procedures. I have been able to see the progression of our larger project, as we enter the second phase, which involves new assays and concepts.
My project has been focusing on testing our hypothesis, which is that proteoglycans, specifically heparan sulfates, interact with the Crb1 protein in retinal degeneration. These past few weeks, I have been using the genetic model to find conclusions to see if the heparan sulfates have an impact on the phenotype of the eye in mice. I have employed several crucial techniques in this regard. Firstly, once the eyes of the mice are fixed first in 4% PFA and Tissue Tek, I have made cryo-sections, which allows us to cut through the different layers of the eye. Once the sections have been dried, I have performed two types of staining: immunohistochemistry and LACE, both of which I have done in the initial few weeks. Ligand and Carbohydrate Engagement Assay (LACE) detects the interaction between receptor and ligand and allows us to see the presence of heparan sulfates throughout the eye.
Immunohistochemistry involves the use of specific antibodies that stain for certain types of cells and regions, such as the photoreceptor cells and the outer limiting membrane. I have learned to increase the efficiency and quality of these two types of staining and there was significant improvement in the specificity of the fluorescent signals. The results further elucidated our hypothesis and allowed us to enter the second phase of our project. In order to analyze the signal and convey the results of the staining, it is necessary to have clear and aesthetic images, taken under the microscope. In the first weeks, my images were blurry and sometimes missed the target region of the eye, due to the cutting of the eye during cryo-sectioning. Since the last report, I have done staining numerous times. I have learned to use the microscope to adjust the slides and various settings on the software used in our lab to create better images. In the final upcoming weeks of the internship, I hope to have excellent pictures to display at the Summer Research Institute Poster Session. I will be presenting the results of these staining in our weekly lab meeting, with the other post-doctoral researchers.
The project has also entered a second phase, turning to focus on the Crb1 protein itself and the possible interaction it may have with heparan sulfates. In order to test our hypothesis, I have been introduced to a new technique, DNA Cloning/ recombinant DNA. By analyzing the genetic sequence of the cDNA of the Crb1 protein, my mentor has identified the regions that we need to target for our hypothesis. Based on this information, we have created primers that will cut the DNA along specific sites. This will allow us to use clone fragments of the Crb1 cDNA that are of interest to create bacterial vectors. These vectors will allow us to see protein expression in a host cell. This aspect of the project has been absolutely fascinating!! Having learned the concept of restriction enzymes and recombinant DNA in previous biology courses, the ability to apply and actually conduct these assays in a molecular lab has been exciting. We are currently testing our primers and are in the initial stages of these functional experiments. In the coming weeks, I hope I can produce good results from these assays and understand and improve the way I carry out the protocol. Furthermore, I will be focusing on synthesizing these two aspects of our project. I want to condense the concepts in a concise manner, in order for me to clearly communicate the theory and results of the project for SRI presentation.
As the internship enters the final weeks, I have been reflecting on the importance of having a supportive and collaborative mentorship. When I first started in the lab last year, I was initially nervous, for it was my first experience in research. I felt overwhelmed at the time, having not taken any biology course in college yet. It was the patience of my mentor that has allowed to grow as a researcher and as a critical thinker. He has taught me the concepts and the techniques and encouraged me, when many of my experiments did not work. He has challenged to me to think as a scientist, which has been invaluable in making my research experience rewarding. This nurturing mentorship is a key concept that we can apply in the field of STEM education. For many, having an individual to guide them can truly change the way they look at STEM. No matter how daunting a challenge can be, having the right motivation and support directly affects our interest in the project at hand. I have been lucky to have positive interactions with the other researchers in the lab as well. They have been sharing their experiences and have helped me understand the challenges in research. I have had the chance to engage with other researchers as well from other research labs in the Department of Ophthalmology in an informal setting. Talking to others has been itself a learning experience and I forward to engaging with the other researchers for the duration of my internship.
In the upcoming weeks, I hope to have my abstract and poster ready for SRI! I look forward to speaking about my project and learning about the other great research projects conducted throughout the summer.