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)-inducible eukaryotic expression vector and to evaluate its regulatory effect in vitro using luciferase reporter gene. Methods: Vector pDC-RULUC, which contains firefly luciferase reporter gene, promoter and mifepristone-inducible system, was constructed by molecular biological methods. A 1.2 kb insulator was inserted to reduce the interference between two transcription units. The vector was verified by PCR, restriction enzyme 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 luciferase 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 recombined 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.