Hamadan University of Medical Sciences Avicenna Journal of Medical Biochemistry 2345-4113 2 2 2014 12 01 Preparation of Preproinsulin Gene Construct Containing the Metallothionein2A (pBINDMTChIns) and Its Expression in NIH3T3 Cell Line and Muscle Tissue of Alloxan Diabetic Rabbits 3 21646 10.17795/ajmb-21646 EN Hossein Piri Bahram Kazemi Iraj Khodadadi Maryam Javadi Mojgan Bandehpour Jamshid Karimi Amir Ziaee Amaneh Koochaki Ali Torabi Mohammad Taghi Goodarzi Journal Article 10.17795/ajmb-21646 2014 07 01 2014 08 03 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.