Construction a plasmid insulin gene vector containing metallothionine IIA (pcDNAMTchhns) and carbohydrate response elements (chER) and its expression in NIT3T3 cell line

Background: Type 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. Objectives: The 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 Methods: Cloning 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. Results: The 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. Conclusions: Gel 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.

Type 1 Diabetes Mellitus; Gene Therapy; Insulin