Submitted: 01 Jul 2014
Revised: 27 Jul 2014
Accepted: 03 Aug 2014
First published online: 30 Sep 2014
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Avicenna J Med Biochem. 2014;2(2): e21646.
doi: 10.17795/ajmb-21646
  Abstract View: 264
  PDF Download: 334
  Full Text View: 192

Research Article

Preparation of Preproinsulin Gene Construct Containing the Metallothionein2A (pBINDMTChIns) and Its Expression in NIH3T3 Cell Line and Muscle Tissue of Alloxan Diabetic Rabbits

Hossein Piri 1,2, Bahram Kazemi 3,4, Iraj Khodadadi 5, Maryam Javadi 6, Mojgan Bandehpour 3,4, Jamshid Karimi 5, Amir Ziaee 7,8, Amaneh Koochaki 3,4, Ali Torabi 9, Mohammad Taghi Goodarzi 10

1 Cellular and Molecular Research Center, Qazvin University of Medical Sciences, Qazvin, IR Iran
2 Department of Biochemistry and Genetics, School of Medicine, Qazvin University of Medical Sciences, Qazvin, IR Iran
3 Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, IR Iran
4 Department of Biotechnology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, IR Iran
5 Department of Biochemistry and Nutrition, School of Medicine, Hamadan University of Medical Sciences, Hamadan, IR Iran
6 Department of Nutrition and Dietary Therapy, School of Medicine, Qazvin University of Medical Sciences, Qazvin, IR Iran
7 Department of Endocrinology, Qazvin University of Medical Sciences, Qazvin, IR Iran
8 Qazvin Metabolic Disease Research Center, Qazvin University of Medical Sciences, Qazvin, IR Iran
9 Influenza Unit, Pasteur Institute of Iran, Tehran, IR Iran
10 0Research Center for Molecular Medicine, Hamadan University of Medical Sciences, Hamadan, IR Iran

Article

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.

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