Construction and identification of a mifepristone inducible eukaryotic expression vector
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Supported by National Natural Science Foundation of China(30571830).

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    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.

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History
  • Received:July 10,2007
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  • Online: April 16,2008
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