Title

Generation of a transgenic beta cell line for study of Glis3 using CRISPR/Cas9 technology

Presenter Information

NancyAnn WebsterFollow

Academic Level at Time of Presentation

Senior

Major

Biology: Pre-pharmacy

Minor

Chemistry

List all Project Mentors & Advisor(s)

Dr. Gary ZeRuth

Presentation Format

Oral Presentation

Abstract/Description

Blood glucose levels are highly regulated to maintain blood glucose homeostasis within an organism. When blood glucose levels are elevated, the pancreatic β cells produce insulin, which signals the cells of the peripheral tissues to take up circulating glucose. Expression of insulin is mediated by a number of transcription factors that associate with the proximal promoter of the insulin gene. Many of these transcription factors are also important in β-cell development and maturation. The Krüppel-like transcription factor, Gli-similar 3 (Glis3) is fundamental for the development of β-cells and is critical for insulin expression in adult tissue. Glis3 has been found to associate with the primary cilia, tiny hair-like sensory organelles extending from the surface of most mammalian cells. It is unclear whether Glis3 is required for ciliogenesis, whether the primary cilia are required for proper Glis3 signaling or whether Glis3 associates with the primary cilia on pancreatic β or ductal cells. There is currently no antibody against Glis3. Thus, most studies of Glis3 have been conducted under overexpression conditions that likely do not represent activity under normal physiological expression levels. To overcome this obstacle and to create a tool that can be used for future studies, we are generating a β cell line that expresses a chimeric Glis3-EGFP fusion protein from the endogenous Glis3 locus using CRISPR/Cas9 technology. This novel cell line will subsequently be used to investigate Glis3 protein localization, posttranslational modifications, and protein-protein interactions under experimental conditions, including chronically elevated levels of glucose and conditions of oxidative stress.

Start Date

16-11-2018 10:30 AM

Fall Scholars Week 2018 Event

Honors College Senior Theses

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Nov 16th, 10:30 AM

Generation of a transgenic beta cell line for study of Glis3 using CRISPR/Cas9 technology

Blood glucose levels are highly regulated to maintain blood glucose homeostasis within an organism. When blood glucose levels are elevated, the pancreatic β cells produce insulin, which signals the cells of the peripheral tissues to take up circulating glucose. Expression of insulin is mediated by a number of transcription factors that associate with the proximal promoter of the insulin gene. Many of these transcription factors are also important in β-cell development and maturation. The Krüppel-like transcription factor, Gli-similar 3 (Glis3) is fundamental for the development of β-cells and is critical for insulin expression in adult tissue. Glis3 has been found to associate with the primary cilia, tiny hair-like sensory organelles extending from the surface of most mammalian cells. It is unclear whether Glis3 is required for ciliogenesis, whether the primary cilia are required for proper Glis3 signaling or whether Glis3 associates with the primary cilia on pancreatic β or ductal cells. There is currently no antibody against Glis3. Thus, most studies of Glis3 have been conducted under overexpression conditions that likely do not represent activity under normal physiological expression levels. To overcome this obstacle and to create a tool that can be used for future studies, we are generating a β cell line that expresses a chimeric Glis3-EGFP fusion protein from the endogenous Glis3 locus using CRISPR/Cas9 technology. This novel cell line will subsequently be used to investigate Glis3 protein localization, posttranslational modifications, and protein-protein interactions under experimental conditions, including chronically elevated levels of glucose and conditions of oxidative stress.