Construction and screening of specific shRNA interference plasmid vector targeting Cyclin E1 gene

Objective To construct a plasmid expression vector coding for the short hairpin RNA (shRNA) targeting Cyclin E1 mRNA and to identify the silencing effect.Methods The sequence of Cyclin E1 mRNA was found in Genebank.After excluding homeology,two plasmid expression vectors coding for shRNA targeting 454 and 708 of Cyclin E1 gene sequence were constructed and the unrelated sequence was used as negative control.Two terminals of shRNA carried BamH Ⅰ and Hind Ⅲ restriction sites and a Sal Ⅰ restriction site was designed at the middle of shRNA fragment.Then shRNA was linked with empty plasmid expression vector with BamH Ⅰ and Hind Ⅲ restriction sites.The recombinant plasmid was identified by polymerase chain reaction (PCR) electrophoresis and sequencing.The inhibition of Cyclin E1mRNA and protein expression was detected by reverse transcription (RT) -PCR and Western blotting in the transfected cells.Results After digestion with Sal Ⅰ,the size of amplified band from plasmid was consistent with the theoretical value and the results of sequencing proved that shRNA was inserted into plasmid successfully.In the control group and negative plasmid transfection group,Cyclin E1 mRNA expression rate was 0.91 ±0.04 and 0.87 ±0.02 respectively,significantly higher than pGenesil-Cyclin E1-shRNA1group 0.11 ± 0.01 and pGenesil-Cyclin E1-shRNA2 group 0.16 ± 0.03 ( P＜0.05 ).Similarly,expression rate of Cyclin E1 protein in the first two groups was 0.89 ± 0.07 and 0.84 ± 0.03 respectively,obviously higher than 0.10 ± 0.06 and 0.14 ± 0.04 in the latter two groups (P＜0.05 ).Conclusion The plasmid expression vector pGenesil-Cyclin E1-shRNA1 targeting Cyclin E1 gene was constructed successfully and the silencing effect was satisfactory. Key words: Cyclin E1 gene;  Short hairpin RNA;  Expression vector;  Plasmid