Construction of pRNAT-U6.1/Neo siRNA System to Knockdown E2F-3 Activity / 天津医药

ABSTRACT

Objective: To construct siRNA plasmid expression vector in order to knockdown E2F-3 activity. Methods: Sixty-four base-pair oligos for hairpin RNA expression, which targeted E2F-3 gene, were chemically synthesized and annealed. The pRNAT-U6.1/Neo vector was linearized with Bam HI and HindⅢ. Finally, the annealed oligos were inserted into the lined pRNAT-U6.1/Neo to construct RNAi plasmid(pRNAT-U6.1-E2F-3/Neo). The reconstructed RNAi plasmids were i-dentified by electrophoresis after digestion with BamHI and Hind Ⅲ, and were confirmed by sequencing analysis. Results: The recombinant pRNAT-U6.1-E2F-3/Neo vector was identified by polymerase chain reaction, and confirmed by sequencing analysis. The results demonstrated that 64 bp had been inserted into the expected site. Furthermore, the insertion sequence was exactly correct and no mutation site was found. Conclusion: The pRNAT-U6.1-E2F-3/Neo RNAi system was constructed successfully. This will facilitate the study of E2F-3 in bladder cancer cell lines.