GLUCOSE RESPONSIVENESS OF A NOVEL SYNTHETIC OVINE SUPER PROMOTER

Abstract

Prior research indicates that constructs containing the endogenous ovine insulin gene promoter have little to no activity in pancreatic beta cells. Our objective was to develop an ovine insulin promoter that is both glucose-responsive and beta-cell specific. To do this, we concatemerized the proximal promoter region of the ovine insulin gene to create a sheep insulin super promoter (SISP). We cloned SISP into two adenovirus constructs with a CMV promoter driving the fluorescent protein ZsGreen and SISP promoting either the fluorescent protein mCherry or luminescent protein luciferase. These constructs were tested in MIN6 cells to determine the promoter's specificity and glucose responsiveness in an insulinoma cell line. Results indicate that SISP had a high infection efficiency in MIN6 and was responsive to higher glucose concentrations. The addition of the antioxidant N-acetyl cysteine had no effect on promoter responsiveness. The next steps for this super promoter include testing in ovine islets and directly comparing its activity to the endogenous ovine insulin promoter. SISP can be used as a tool for beta cell isolation, monitoring glucose-stimulated insulin secretion, and targeting gene expression in beta cells.