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The Interference Effect of Vector-based siRNA on Survivin Gene of Human Breast Cancer SKBr-3 Cells
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Abstract:
Objective:To construct the eukaryotic expression vector for RNA interferencing human survivin gene and detect its interference effect in human breast cancer cell line(SKBr-3). Methods: Two target gene segments were synthesized and cloned into pSUPER vector respectively to construct two recombinant eukaryotic expression vectors: pSUPER-S1 and pSUPER-S2. The two recombinant vectors were identified by enzyme digestion analysis and DNA sequencing. Then SKBr-3 cells were transfected with pSUPER-S1 or pSUPER-S2, together with pCDNA3 plasmid, and subjected to G418 selection. In G418-resistant cells, the interference effect was detected by RT-PCR, Western blot and immunocytochemical staining. Results: Enzyme digestion analysis and DNA sequencing showed that the target segments were cloned into pSUPER vector respectively. The results of RT-PCR, Western blot and immunocytochemical staining indicated that both vectors could knock down the transcription and expression of survivin gene, and that pSUPER-S1 had better interference effect than pSUPER-S2. Conclusion:The transcription and expression of survivin gene were inhibited effectively by the constructed RNAi eukaryotic expression vectors in the breast cancer cells.
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