Construction and identification of a mifepristone inducible eukaryotic expression vector / 第二军医大学学报

ABSTRACT

Objective:To construct a mifepristone(an oral nontoxic chemical)-indueible eukaryotic expression vector and to evaluate its regulatory effect in vitro using luciferase reporter gene.Methods:Vector pDC-RULUC,which contains firefly lucif- erase reporter gene,promoter and mifepristone-inducible system,was constructed by molecular biological methods.A 1.2 kb in sulator was inserted to reduce the interference between two transcription units.The vector was verified by PCR,restriction en- zyme digestion,and sequencing,pDC-RULUC was used to transfect SW620 cells using Lipofectamine2000.Cells transfected with pGL3-Control and pGL3-Basic were used as positive and negative controls,respectively.Cotransfectant with pRL-TK renilla lu- ciferase reporter vector was used as internal control.Cells of experimental group were incubated for 48 h in presence of different concentrations of mifepristone after transfection and were harvested for luciferase assay by using the Dual-Luciferase Reporter Assay System.Half of the wells were replaced with fresh medium and were measured after another 48 h.Results:The recom- bined plasmid vector was identified by digestion with different enzyme restrictions,PCR and sequencing analysis.The relative activity increased with the increase of mifepristone concentration.When the concentration of mifepristone reached 1?10~(-6)mol/ L,the relative activity increased to approximately 50 folds of the original.No significant luciferase activity was detected when the mifepristone was removed.Conclusion:We have successfully established mifepristone-regulated eukaryotic expression vector, which can be used for controllable gene expression in vitro,providing a way for gene regulation and gene therapy.