Author/Authors :
Piri, Hossein hamadan university of medical sciences - School of Medicine - Department of Biochemistry and Nutrition, ايران , Kazemi, Bahram shahid beheshti university of medical sciences - Cellular and Molecular Biology Research Center, تهران, ايران , Kazemi, Bahram shahid beheshti university of medical sciences - Faculty of Medicine - Biotechnology Department, تهران, ايران , Rezaei, Mohsen hamadan university of medical sciences - School of Medicine - Department of Biochemistry and Nutrition, ايران , Bandehpour, Mojgan shahid beheshti university - Cellular and Molecular Biology Research Center, تهران, ايران , Bandehpour, Mojgan shahid beheshti university of medical sciences - Faculty of Medicine - Biotechnology Department, تهران, ايران , Khodadadi, Iraj hamadan university of medical sciences - School of Medicine - Department of Biochemistry and Nutrition, ايران , Hassanzadeh, Taghi hamadan university of medical sciences - School of Medicine - Department of Biochemistry and Nutrition, ايران , Karimi, Jamshid hamadan university of medical sciences - School of Medicine - Department of Biochemistry and Nutrition, ايران , Yarian, Fatemeh shahid beheshti university - Cellular and Molecular Biology Research Center, تهران, ايران , Yarian, Fatemeh shahid beheshti university of medical sciences - Faculty of Medicine - Biotechnology Department, تهران, ايران , Peirovi, Habibollah shahid beheshti university of medical sciences - Nano Medicine and Tissue Engineering Research Center, تهران, ايران , Tavakoli, Amir Hossein tehran university of medical sciences tums - Imam Khomeini Hospital Complex - Iranian Tissue Bank Research and Preparation Center, تهران, ايران , Goodarzi, Mohammad Taghi hamadan university of medical sciences - Research Center for Molecular Medicine, ايران
Abstract :
Background: Type 1 diabetes mellitus is one of the metabolic diseases that cause insulinproducing pancreatic β cells be destroyed by immune system self-reactive T cells. Recently, new treatment methods have been developed including use of the stem cells, β 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.
