Construction and Evaluation of Short Hairpin RNAs for Knockdown of Metadherin  mRNA

Farahnaz   Zare; Sedigheh   Sharifzadeh; Abbas  Behzad-Behbahani; Gholamreza   Rafiei Dehbidi; Zahra  Yousefi; Reza  Ranjbaran; Noorossadat  Seyyedi

doi:10.18502/ajmb.v13i4.7207

Farahnaz Zare Division of Medical Biotechnology, Department of Medical Laboratory Sciences, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
Sedigheh Sharifzadeh Diagnostic Laboratory Sciences and Technology Research Center, Faculty of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
Abbas Behzad-Behbahani Division of Medical Biotechnology, Department of Medical Laboratory Sciences, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
Gholamreza Rafiei Dehbidi Diagnostic Laboratory Sciences and Technology Research Center, Faculty of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
Zahra Yousefi School of Allied Medical Sciences, Shahroud University of Medical Sciences, Shahroud, Iran
Reza Ranjbaran Diagnostic Laboratory Sciences and Technology Research Center, Faculty of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
Noorossadat Seyyedi Division of Medical Biotechnology, Department of Medical Laboratory Sciences, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran

DOI: https://doi.org/10.18502/ajmb.v13i4.7207

Keywords: Apoptosis, Gene expression, Gene silencing, Jurkat cells, RNA

Abstract

Background: Short hairpin RNA (shRNA) has proven to be a powerful tool to study genes’ function through RNA interference mechanism. Three different methods have been used in previous studies to produce shRNA expression vectors including oligonucleotide-based cloning, polymerase chain reaction (PCR)-based cloning, and primer extension PCR approaches. The aim of this study was designing a reliable and simple method according to the primer extension strategy for constructing four shRNA vectors in order to target different regions of Metadherin (MTDH) mRNA in human leukemic cell line Jurkat.

Methods: Oligonucleotides for construction of four shRNA vectors were designed, synthesized and fused to U6 promoter. Each U6-shRNA cassette was cloned into a pGFP-V-RS vector. MTDH shRNAs were transfected into the Jurkat cell line by using the electroporation method. The ability of shRNAs to knock down MTDH mRNA was analyzed through qRT-PCR. Apoptosis assay was used to evaluate the effect of down regulation of MTDH expression on cell integrity.

Results: A significant reduction (about 80%) in the expression levels of MTDH mRNA and an increase in the percentages of apoptotic cells (about 20%) were observed in the test group in comparison with control.

Conclusion: MTDH shRNA constructs effectively inhibited gene expression. However, simplicity and inexpensiveness of the method were additional advantages for its application.