Construction and Identification of pH1-EphB4-shRNA Expression Vector

Objective To construct short hairpin RNA(shRNA) eukaryotic expression vector,targeting gene EphB4 which may play an important role in glioma pathogenesis.Methods A specific DNA oligonucleotide targeting gene EphB4 on appropriate site was synthesized and inserted into Bbs I linearized psiRNA-H1 vector.The sequence of the plasmid was identified by DNA sequencer and restriction endonuclease digestion.The recombinant plasmid was transfected into the glioma cell lines U251 by lipofection.After transfected 48 hours,the inhibition of EphB4 mRNA expression was detected by RT-PCR.Results It was verified that the specific DNA oligonucleotide was cloned into the vector successfully,and the expression of EphB4 mRNA in U251 cells was significantly reduced by 60% after transfecting the recombinant plasmid,compared to the controls.Conclusion It indicates the shRNA eukaryotic expression vector has been successfully established which can inhibit the expression of EphB4 mRNA,and those provides the precondition for the further study of EphB4 in glioma pathogenesis.