Miranda Jefferds
Pronouns: she/her
Research Mentor(s): Adelaide Tovar
Research Mentor School/College/Department: Computational Medicine & Bioinformatics / Medicine
Program:
Authors: Miranda Jefferds, Adelaide Tovar, Stephen Parker, Jacob Kitzman
Session: Session 6: 3:40 pm – 4:30 pm
Poster: 71
Abstract
Type II diabetes mellitus (T2D) is a condition in which your body has insulin resistance, lack of insulin, and/or high blood sugar. It affects much of the population and susceptibility to this disease is significantly impacted by your genetic makeup. I aim to identify novel genes that are crucial for insulin production and glucose regulation while also expanding on what we already know about the pathways and processes that are involved in T2D. For this project, I have focused on the transcription factor gene RFX6, which is known to cause monogenic, early life forms of diabetes. Because this gene plays a large role in the processes that can lead to T2D, I hypothesize that knocking it out will yield many phenotypic changes. To accomplish this, I designed guide RNAs targeting this gene, cloned them into plasmids that we used to generate lentivirus. I infected the human beta cell lines EndoC-BH1 and EndoC-BH3 with these viruses to generate cells with modified or knocked out RFX6. I performed genotyping PCR and sent products for sequencing to see changes in sequences and target regions between the WT and KO lines. I also used quantitative PCR (qPCR) to see gene expression profiles in both sets of cells and then tested these modified lines through a variety of assays. This included RNA-seq to see gene expression changes throughout the entire genome, Western blots to examine protein expression of RFX6, and glucose-stimulated insulin secretion (GSIS) to discern if the cells will respond normally to an induced increase in glucose by secreting insulin. I plan to relate these results to the bigger picture of identifying how we can mitigate diabetes – if we can figure out which gene mutations lead to having Type II diabetes, gene modifying drugs or therapies can be created to try to reverse the growing onset of diabetes.
