title: Construction of expressing vectors including melanoma differentiation-associated gene-7 (mda-7) fused with the RGD sequences for better tumor targeting.
abstract: OBJECTIVE
  Up to now, many researches have been performed to improve the antitumoral effect of melanoma differentiation-associated gene-7 (mda-7) protein. The purpose of our research was to construct 3 expression vectors producing mda-7 in fusion with RGD (Arginine-Glycine-Aspartic acid) peptide and evaluate their expression.
  
  
  METHODS
  mda-7 gene with two different RGD sequences was amplified by PCR then was cloned by TA-cloning system. The colonies including these genes were selected by blue-white screening, colony PCR, and sequencing, respectively. Afterward, the genes were sub-cloned into the expression vector following confirmation by colony PCR and sequencing. In addition, these constructs were transfected into 293 and Huh-7 cells for further expression analysis. The mda-7 gene expression was evaluated by RT-PCR and IF (immunofluorescence assay). DNA laddering test and trypan blue exclusion assays were performed to screen cytotoxicity of prepared plasmids.
  
  
  RESULTS
  Three different mda-7 genes with terminal RGD peptide were cloned correctly into the expression vectors and their expression was confirmed to be suitable by RT-PCR and IF assay. It was shown that expressions were limited to those transfected, GFP shining cells. No significant cytotoxicity was observed by simple assays in all plasmid treated cells. In expressing cells, all forms of mda-7 protein were localized mainly around ER prenuclear compartment while GFP protein was distributed evenly among them.
  
  
  CONCLUSIONS
  Theoretically RGD tagged mda-7 would be able to induce apoptosis with more specificity and stronger than the standard one, therefore, these new constructs may have the potential for further researches.
authors.affiliation: Gastroenterohepatology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.
authors.forename: Mahboobeh
authors.initials: M
authors.lastname: Khodadad
authors.affiliation: Gastroenterohepatology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.
authors.forename: Seyed Younes
authors.initials: SY
authors.lastname: Hosseini
authors.affiliation: Gastroenterohepatology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.
authors.forename: Fatemeh
authors.initials: F
authors.lastname: Shenavar
authors.affiliation: Cancer Immunology Research Group, Shiraz Institute for Cancer Research, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
authors.forename: Nasrollah
authors.initials: N
authors.lastname: Erfani
authors.affiliation: Gastroenterohepatology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran ; Sciences Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran.
authors.forename: Samaneh
authors.initials: S
authors.lastname: Bina
authors.affiliation: Sciences Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran.
authors.forename: Shahin
authors.initials: S
authors.lastname: Ahmadian
authors.affiliation: Gastroenterohepatology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.
authors.forename: Mohammad-Reza
authors.initials: MR
authors.lastname: Fattahi
authors.affiliation: Department Of Biochemistry, Payame Noor University, Tehran Shargh Branch, Tehran, Iran.
authors.forename: Reza
authors.initials: R
authors.lastname: Hajhosseini
keywords: Mda-7; RGD peptide; Tumor targeting