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

authors:

avatar Hossein Piri 1 , avatar Bahram Kazemi 3 , avatar Mohsen Rezaei 1 , avatar Mojgan Bandehpour 3 , avatar Iraj Khodadadi 1 , avatar Taghi Hassanzadeh 1 , avatar Jamshid Karimi 1 , avatar Fatemeh Yarian 3 , avatar Habibollah Peirovi 4 , avatar Amir Hossein Tavakoli 5 , avatar Mohammad Taghi Goodarzi 6 , *

Department of Biochemistry and Nutrition, School of Medicine, Hamadan University of Medical Science, IR Iran
Biotechnology Department, Faculty of Medicine, Shahid Beheshti University of Medical Science, IR Iran
Nano Medicine and Tissue Engineering Research Center- Shahid Beheshti University of medical sciences, IR Iran
Iranian Tissue Bank Research and Preparation Center, Imam Khomeini Hospital Complex, Tehran University of Medical Science, IR Iran
Research Center for Molecular Medicine, Hamadan University of Medical Science, [email protected], IR Iran

how to cite: Piri H, Kazemi B, Rezaei M, Bandehpour M, Khodadadi I, et al. Construction of Plasmid Insulin Gene Vector Containing Metallothionein IIA (pcDNAMTChIns) and Carbohydrate Response Element (ChoRE), and Its Expression in NIH3T3 Cell Line. Int J Endocrinol Metab.10(3): 543-547. doi: 10.5812/ijem.4540.

Abstract

Background:

Type 1 diabetes mellitus is one of the metabolic diseases that cause insulin-producing pancreatic cells be destroyed by immune system self-reactive T cells. Recently, new treatment methods have been developed including use of the stem cells, 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.

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