Construction and identification of siRNA expression vectors for silencing PTTG gene

Objective To construct the specific high efficiency small interfering RNA(siRNA)expression vector that can silence PTTG gene.Methods Using vector based RNA interference technique,vectors were constructed to transcribe the functional short hairpinRNA(shRNA) specially targeting PTTG.The vectors were used to transfect AtT20 cells by lipofectamine2000 reagent.And the cells were divided into five groups:normal control,negative group and three siRNA interfering groups(pGenesil2-PTTG siRNA1,pGenesil2-PTTG siRNA2 and pGenesil2-PTTG siRNA3).The expression levels of mRNA and protein of PTTG were analyzed by RT-PCR and Western blotting methods.Results By restriction endonuclease and DNA sequencing analyzing,eukaryotic expression plasmid of PTTG was successfully constructed.PTTG mRNA and its protein level in transfected AtT20 cells with PTTG RNAi plasmid decreased significantly compared with the normal control group(P0.01),and the transfection rate reached approximately 75%.Conclusion pGenesil2-PTTG siRNA vectors were successfully constructed,and they can silence the PTTG gene in AtT20 cells with high efficiency.