title: Construction of Plasmid Insulin Gene Vector Containing Metallothionein IIA (pcDNAMTChIns) and Carbohydrate Response Element (ChoRE), and Its Expression in NIH3T3 Cell Line.
abstract: 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. Recent-ly, 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.
  
  
  OBJECTIVE
  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.
  
  
  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 extract-ed 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 preproinsu-lin gene and expression was confirmed by Reverse Transcriptase PCR and Western Blot-ting Techniques.
  
  
  CONCLUSIONS
  Gel electrophoresis of PCR products confirmed that cloning was per-formed correctly. The expression of preproinsulin gene in recombinant plasmid in NI-H3T3 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.
authors.affiliation: Department of Biochemistry and Nutrition, School of Medicine, Hamadan University of Medical Science, Hamadan, IR Iran.
authors.forename: Hossein
authors.initials: H
authors.lastname: Piri
authors.forename: Bahram
authors.initials: B
authors.lastname: Kazemi
authors.forename: Mohsen
authors.initials: M
authors.lastname: Rezaei
authors.forename: Mojgan
authors.initials: M
authors.lastname: Bandehpour
authors.forename: Iraj
authors.initials: I
authors.lastname: Khodadadi
authors.forename: Taghi
authors.initials: T
authors.lastname: Hassanzadeh
authors.forename: Jamshid
authors.initials: J
authors.lastname: Karimi
authors.forename: Fatemeh
authors.initials: F
authors.lastname: Yarian
authors.forename: Habibollah
authors.initials: H
authors.lastname: Peirovi
authors.forename: Amir Hossein
authors.initials: AH
authors.lastname: Tavakoli
authors.forename: Mohammad Taghi
authors.initials: MT
authors.lastname: Goodarzi
keywords: Gene Therapy; Insulin; Type 1 Diabetes Mellitus