Construction of Eukaryotic Vector Expressing Three shRNAs of GSK3β Gene

Objective：To explore the feasibility of selective inhibiting GSK3β expression using GSK3β short hairpin RNA (shRNA) interference. Methods：Three 19bp reverse repeated motifs targeted to GSK3β gene were synthesized and cloned into eukaryotic expression plasmid pshRNA -U3 containing three U6 shRNA promoter and termination signal of RNA polymerase. The recombinant plasmids pshRNA-U3/GSK3β and pshRNA-U3/con were transfected into OV2008 cells by lipofectamine reagent，respectively.The alteration of GSK3β expression was examined by real time-PCR and Western blot. Results：It was verified by partial nucleotide sequencing that the constructed eukaryotic vector expressing three shRNA of GSK3β was correct.OV2008 cellstransfected by pshRNA-U3/GSK3β dramatically down-regulated the expression of GSK3β mRNA and protein compared with untransfected and control cells. Conclusion：The shRNA can efficiently suppress expression of GSK3β in OV2008 cells. The results of the study lay the foundation for further studying on biological functions and potential application of GSK3β.