Gabriella Bloss
Pronouns: she/her
Research Mentor(s): Adelaide Tovar
Research Mentor School/College/Department: Computational Medicine & Bioinformatics / Medicine
Program:
Authors: Gabriella Bloss, Adelaide Tovar, Jacob Kitzman, Stephen Parker
Session: Session 6: 3:40 pm – 4:30 pm
Poster: 70
Abstract
This study investigates the functional impact of disrupting the transcription factor (TF) hepatocyte nuclear factor-1 beta (HNF1B). This TF is associated with the development of early-onset diabetes, also known as maturity-onset diabetes of the young (MODY). MODY encompasses rare forms of diabetes caused by mutations in specific genes, one of which is HNF1B. I designed CRISPR guide RNAs (gRNAs) to specifically target the DNA binding domain of my target gene. I then cloned these gRNAs into lentiCRISPRv2 plasmid backbones, transformed bacteria, and used them to generate two different lentiCRISPR viruses. I infected two types of immortalized human beta cell lines, EndoC-BH1 (BH1) and EndoC-BH3 (BH3), with each virus separately and in combination to create six knockout lines and two wild-type lines. After the cells underwent antibiotic selection to enrich for edited cells, I performed several tests to confirm the success of the editing. These tests included genotyping PCR and Sanger sequencing, qPCR, and RNA-sequencing. For genotyping PCR, I ran a gel which showed differences in sequencing between the knockout and wild-type lines. We sent these PCR products for Sanger sequencing to gain a better idea of the extent and success of these edits. Then, I conducted qPCR to examine gene expression, comparing the knockout lines to the wild type. The test revealed decreased gene expression in the BH3 lines but surprisingly increased gene expression in the BH1 lines. Using the combined data from Sanger sequencing and qPCR to select suitable lines, we generated whole-transcriptome RNA-seq libraries for the wild-type and double knockout lines on both backgrounds. Once we confirm the success of the lentiCRISPRv2 viruses in knocking out HNF1B, these cell lines can serve as models for patients with this specific subset of MODY to understand the full implications of this specific mutation on the development and progression of diabetes.
