Construction of Plasmid Insulin Gene Vector Containing Metallothionein IIA (pcDNAMTChIns) and Carbohydrate Response Element (ChoRE), and Its Expression in NIH3T3 Cell Line

Abstract

BackgroundType 1 diabetes mellitus is one of the metabolic diseases that cause insulin-producing pancreatic ß cells be destroyed by immune system self-reactive T cells. Recent­ly, new treatment methods have been developed including use of the stem cells, ß islet cells transplantation and gene therapy by viral and non-viral gene constructs.ObjectivesThe aim of this project was preparing the non-viral vector containing the glucose inducible insulin gene and using it in the NIH3T3 cell line.Materials and MethodsCloning was carried out by standard methods. Total RNA was extracted from pancreatic tissue, RNA was converted to cDNA using RT-PCR reaction and preproinsulin gene was amplified using specific primers. PNMTCH plasmid was extract­ed and digested by NotI, HindIII, and MTIIA and ChoRE genes were purified and cloned into pcDNA3.1 (-) plasmid and named pcDNAMTCh. Finally, the preproinsulin genes were cloned into pcDNA3.1 (-) plasmid and pcDNAMTChIns was built.ResultsThe cloned gene constructs were evaluated by restriction enzyme digestion and RT-PCR. The NIH3T3 cells were transfected by plasmid naked DNA containing preproinsu­lin gene and expression was confirmed by Reverse Transcriptase PCR and Western Blot­ting Techniques.ConclusionsGel electrophoresis of PCR products confirmed that cloning was per­formed correctly. The expression of preproinsulin gene in recombinant plasmid in NI­H3T3 cell line was observed for the first time. The findings in this study can be the basis of further research on diabetes mellitus type 1 gene therapy on animals.