Establishment of stable RNAi system in human cell lines and cDNA libraries

• Main Authors: Chien-chih Chen, 陳建志
• Other Authors: Jinghua T. Chang, Ph. D.
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/92738858337152237078

碩士 === 中山醫學大學 === 毒理學研究所 === 91 === Abstract RNA interference (RNAi) is referred to a phenomenon of gene-specific silencing triggered by double-stranded RNA. RNAi is the process of sequence-specific, post-transcriptional gene silencing. This mechanism has been found in animals, plants and fungi. However, it was not able to apply to mammalian cell cluture and mammals. Recently, chemically synthesized 21-nt siRNA duplex with symmetric 2-nt overhangs at its 3'' ends triggered RNAi in mammalian culture cells successfully. Further development of a vector-based RNAi technology results in stable suppressing specific gene expression in mammalian cells. these DNA vectors contain either H1 promoter or U6 promoter. After transfection, these promoters express shRNA (small hairpin RNA) via RNA polymerase III. In this project, a human U6 fragment, from —305 to +1, was PCR amplified as a promoter to express p53RNAi DNA fragment. After insertion of U6-p53RNAi DNA fragment into pcDNA3.1/CMV- vector, this construct is transfected into A549 cells. The G418 resistant stable clones showed strong activity in repression the expression level of p53 via real-time PCR and Western analyses.