Destruction of the neural retina in mammals currently leads to irreversible blindness. Unlike humans, newts possess the ability to regenerate a functional neural retina from their retinal pigmented epithelium (RPE), a cell type that lies behind the neural retina. We will develop a method to individually regulate genes by activating and repressing specifically targeted genes. This method will then be used to test and determine what transition factors are required for retinal regeneration. If we can verify transcription factors that are important in the differentiation of neural retina in newts, we can attempt to adapt this regeneration pathway for mammals. We will develop a plasmid construct that uses the CRISPR-Cas9 system in conjunction with a shortened or dead guide RNA (dgRNA) to reprogram cell differentiation. This construct will additionally contain a transcriptional activator and repressor that will work with the Cas9 protein to allow targeted genes to be turned on or off. This plasmid construct is first inserted into a human induced pluripotent stem cell (hiPSC) line that was engineered to express different fluorescent proteins to correlate with different tissues in the neural retina. The hiPSC line allows the construct’s ability to regenerate a cell’s fate to be verified. Next, these hiPSC cells will be differentiated into RPE cells and transfected with gene-specific dgRNAs that will target sequences to activate the genes expressed in neural retina and repress the genes expressed in RPE. This technique will help to determine the key transcription factors required to redirect RPE into neural retina cells and help to develop a possible cure to permanent eye damage. This project has heightened my interest in research and has the potential to benefit many people which aligns with my interest in pursuing a career in the medical field.
Author: Dana Kapcio
Faculty Advisors: Dr. Michael Robinson and Tycho Jaquish, Department of Biology
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