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Construction of RNAi Vectors for RNA Interference of Bloom Helicase Gene in Human Prostate Cancer PC3 Cells
Luo Mutan1,2, Xu Houqiang2*, Liu Zhongwei2,3, Duan Zhiqiang2, Zhao Jiafu1,2, Wu Ping1, Chen Fu1
1College of Life Science, Guizhou University, Guiyang 550025, China;
2College of Animal Science,Key Laboratory of Animal Genetics, Breeding and Production in the Pleteau Mountains Region, Ministry of Education,Guizhou University, Guiyang 550025, China;
3College of Agriculture, Guizhou University, Guiyang 550025, China
2College of Animal Science,Key Laboratory of Animal Genetics, Breeding and Production in the Pleteau Mountains Region, Ministry of Education,Guizhou University, Guiyang 550025, China;
3College of Agriculture, Guizhou University, Guiyang 550025, China
Abstract: RNA interference (RNAi) technology was used in this research for the sake of down-regulating the expression of Bloom helicase in human prostate cancer PC3 cells. At the early stage of the experiment， vectors capable of producing short hairpin RNA (shRNA) molecule for Bloom helicase were constructed using the mammalian expression plasmid vector CMV-copGFP-T2A-Puro-H1-mcs. After the vectors were successfully constructed and confirmed by DNA sequencing， the PC3 cells were transfected with the constructed positive and negative RNAi vectors. Real-time fluorescence quantitative PCR (RTFQ PCR) and Western blot were used to examine the mRNA and protein levels in the transfected PC3 cells. The results indicated that the Bloom helicase mRNA and protein level decreased significantly after tranfection of the positive vectors in PC3 cells， while in negative plasmid transfected PC3 cells， Bloom helicase expression remained the same level as the normal cells. These data suggested that Bloom helicase expression could be inhibited by shRNA transfectants in PC3 cells at mRNA and protein levels.
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