RNAi-2006诺贝尔生理学奖或医学奖.pptVIP

最初由Harvard医学院教授构建并成功运用于knock down expression of cdk-2 and lamin A/C in HeLa, H1299, U-2 OS and C-33A (cdk-2 only) 细胞中 Protocol与环状结构模式图 most of the groups developed plasmids with a sequence encoding a small hairpin RNA (shRNA) under the control of an RNA Polymerase III (Pol III) promoter. The U6 and H1 Pol III promoters were chosen because Pol III initiates synthesis at a defined distance from these promoters and terminates when a string of 4-5 uridines is encountered. When transfected into mammalian cells, siRNA expression plasmids and have been shown to reduce the levels of both exogenous and endogenous gene products。 To use the vector, one needs to first linearize it with Apa I and EcoR I and design an appropriate ~55 bp insert sequence. (1) a linearized and purified vector ready for ligation; (2) a GAPDH-specific siRNA insert that can be used as a positive control; (3) a circular, negative control pSilencer Vector that expresses an siRNA with limited homology to any known sequences in the human, mouse, and rat genomes; and (4) 1X DNA Annealing solution to prepare DNA oligonucleotides for ligation. pSilencer 3.0-H1 features the H1 RNA promoter (H1 RNA is a component of RNase P). The pSilencer 2.0-U6 and 3.0-H1 siRNA expression vectors are linearized with BamH I and Hind III, which leave 3 overhangs that facilitate directional cloning. The linearized vectors are purified to eliminate re-ligation of the vector with the digested insert. The 3 overhangs are the same for both the pSilencer 2.0-U6 and the 3.0-H1 Vectors (but are distinct from the linearized pSilencer 1.0-U6 Vector), making it possible to subclone the same siRNA insert into either pSilencer 2.0-U6 or 3.0-H1. P o w e r B a r 中国专业PPT设计交流论坛 Gene Company Limited 基 因 有 限 公 司 内容摘要 一、获奖者简介 二、RNAi现象的发现 三、RNAi现象的分子机理 四、RNAi的应用 一、获奖者简介 安德鲁·法尔(Andrew Z.Fire），美国科学家、生物医学家。1959年出生于美国，1983年获美国麻省理工学院生物学博士学位，现任斯坦福医学院病理学和遗传学教授。主要研究方向为细胞和组织对遗传改变的反应机制。 克雷格·梅洛（Craig C. Mello),1960年出生，1990年获得哈佛大学生物学博士学位，现任美国马萨