In the first two weeks of my internship, I have been honing wet-lab techniques and understanding the theory and goals for my project. Although I have been working in the Zhang lab at the Columbia University Medical Center part-time since June 2016, working full-time is a new experience that is rewarding, yet challenging. The internship requires me to recall and apply the concepts I have learned in previous biology courses, such as cell signaling pathways, genetics, and basic biochemistry, in a new, dynamic context. This application of class material to real-life research has been a great experience thus far.
Dr. Zhang’s lab focuses primarily on the mechanism of the fibroblast growth factor (FGF) signaling pathway on retinal development, as well as the role of proteoglycans in the retina. Research is conducted on the interactions between various proteins and other signaling pathways with FGF-signaling. The impact of these interactions is analyzed at different stages of development of the neural retina, starting at the embryonic level. This will help us understand the genetic basis for various diseases of the eye and possibly find cures or treatment for these diseases in the future.
My project focuses on the interaction of proteoglycans and the crumbs homolog 1 (Crb1) protein in Leber’s congenital amaurosis (LCA) and some forms of Retinitis pigmentosa (RP), both of which occur at an early-onset. LCA and RP are genetic degenerative disorders that cause progressive destruction of the photoreceptor cells in the retina. Starting from a young age, an individual who has inherited this disease progressively loses vision from the peripheral side of the eye to the entire eye. Prior research has demonstrated that several mutations in the Crb1 gene are associated with LCA and RP. The project that my mentor, a post-doctoral researcher, and I are working on seeks to understand if and how proteoglycans - specifically, heparan sulfates -interact with the Crb1 protein in these disorders. I have, thus far, been trying to analyze the phenotypes for various genes that are known to be involved in RP by using mouse models to look for any similarities and differences. Furthermore, I have been reading literature and trying to understand the theoretical basis for my project. This literature review has been essential in seeing how my project ties into previous research and the new avenue in which our project is taking the field of ophthalmology. Within the duration of the summer, I wish to produce conclusive results that provide more information about our hypothesis. I will be presenting my results on a weekly basis in our lab’s group meeting along with the other post-docs in the lab.
I have learned a wide array of wet-lab techniques in the lab, thus far. Firstly, I genotype newly born litters, through the process of polymerase chain reaction (PCR) and seeing the results on gel electrophoresis. This is crucial in understanding whether the phenotypes we see in the mice are in correspondence with our prediction based on the genotype. Following PCR and gel electrophoresis, I have learned to do cryo-sectioning. My mentor fixes the eyes that are removed from mice in a solution overnight, which makes a block with the eye in the center. In order to analyze these eyes, it is necessary to make sections of these blocks on a microscopic slide. I have learned to do this through cryo-sectioning. It was initially a bit hard to get all the sections to have equal parts of the retina. By repeating the cryo-sectioning on more blocks, I have been improving my technique. Thirdly, I have been conducting various staining on these sections of the eye to understand the phenotypes that can be seen. For instance, I have conducted LACE staining, which shows the distribution of heparan sulfates in the eye. Furthermore, I have done numerous immunohistochemistry stains using several different antibodies, which stain for different types of retinal cells and structures, such as the photoreceptors and the outer limiting-membrane. I hope to improve on these techniques in the coming weeks and produce clear and precise images of the sections of the eye.
The greatest challenge of working in a research lab has been improvising when the assays do not go in the direction you had hoped for. For instance, in many instances, the PCR and some of the staining did not work for me. This was disheartening initially because of the many steps in the experimental assays, especially for staining, which takes two days to see the results. However, from this comes the greatest reward of working in the research lab. I rationalize the possible causes of why the assay did not work and repeat the protocol with the new modifications. This allows me to understand how research works in a real-life setting. It does not go perfectly all the time, but it is part of the process in becoming a better critical-thinker and scientist. This is a lesson that I truly value. My experience in STEM research has been rewarding and I am excited to see what the rest of the summer brings!