Abstract

Background: Diabetes mellitus type 1, formerly called insulin-dependent diabetes, is one of the autoimmune diseases where insulinproducing cells are destroyed by autoimmune response via T cells. The new approaches in treatment of diabetes are using the stem cells, cell transplantation of islet β cell, gene transfer by virus based plasmids, and non-viral gene constructs. Objectives: The purpose of this study was to construct glucose inducible insulin gene plasmid and use it in the muscle tissue of the rabbit. Materials and Methods: To achieve this goal, the preproinsulin, metallothionein2A promoter and the response element to carbohydrate genes were cloned into pBIND plasmid by standard cloning methods, to construct pBINDMTChIns. The gene cloning products were confirmed by the polymerase chain reaction (PCR) and restriction enzyme digestion template. The recombinant plasmid, containing the preproinsulin gene, was transferred into NIH3T3 cells and insulin gene expression was evaluated by reverse transcriptase PCR and western blotting techniques. Plasmid naked DNA containing the preproinsulin gene was injected into the rabbits’ thigh muscles, and its expression was confirmed by western blotting method. Results: This study shows the prepared gene construct is inducible by glucose. Gene expression of preproinsulin was observed in muscle tissue of rabbits. Conclusions: These finding indicated that research in diabetes mellitus gene therapy could be performed on larger animals.

Highlights

  • Diabetes mellitus type 1, formerly called insulin-dependent diabetes, is one of the autoimmune diseases where insulinproducing cells are destroyed by autoimmune response via T cells

  • Considering that diabetes mellitus is increasing in the world, and the classical treatment cannot improve disease appropriately; we designed pBIND eukaryotic vector consisting of the metallothionein2A promoter, the carbohydrates response element, and human preproinsulin gene (Gen Bank: JQ951950.1)

  • Free preproinsulin gene pBIND plasmid was injected to the second group of animals; endotoxin-free sterile PBS was injected to the third group and pBINDMTChIns plasmid was injected to the fourth group

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Summary

Introduction

Diabetes mellitus type 1, formerly called insulin-dependent diabetes, is one of the autoimmune diseases where insulinproducing cells are destroyed by autoimmune response via T cells. The new approaches in treatment of diabetes are using the stem cells, cell transplantation of islet β cell, gene transfer by virus based plasmids, and non-viral gene constructs. Objectives: The purpose of this study was to construct glucose inducible insulin gene plasmid and use it in the muscle tissue of the rabbit. Various procedures (such as pancreas transplantation, using stem cells, or islet cells) have been conducted to induce better results with fewer side effects. These methods can cause serious problems like risks related to surgery, lack of pancreas, graft rejection of the pancreas, and high cost [6, 7]. Vectors containing glucose 6-phosphatase and GLUT-2 promoter have already been used in cell lines and animal tissues of mice and rat [14, 15]

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