Construction and Evaluation of Short Hairpin Rnas For Knockdown of Metadherinmrna

Short hairpin rna (shrna) has proven to be a powerful tool to studygenes’ function through rna interference mechanism. three different methods havebeen used in previous studies to produce shrna expression vectors including oligonucleotide-based cloning, polymerase chain reaction (pcr)-based cloning, and primerextension pcr approaches. the aim of this study was designing a reliable and simplemethod according to the primer extension strategy for constructing four shrna vectorsin order to target different regions of metadherin (mtdh) mrna in human leukemiccell line jurkat.methods: oligonucleotides for construction of four shrna vectors were designed, synthesizedand fused to u6 promoter. each u6-shrna cassette was cloned into a pgfpv-rs vector. mtdh shrnas were transfected into the jurkat cell line by using theelectroporation method. the ability of shrnas to knock down mtdh mrna was analyzedthrough qrt-pcr. apoptosis assay was used to evaluate the effect of downregulation of mtdh expression on cell integrity.results: a significant reduction (about 80%) in the expression levels of mtdh mrnaand an increase in the percentages of apoptotic cells (about 20%) were observed inthe 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.