《发育生物学》第3版课件003-研究方法.pptVIP

* RNAi 研 究 史 1990，Rich Jorgensen 在矮牵牛花中发现转基因沉默 1995，researchers Guo 和Kemphues 注入反义RNA及正义链RNA都能抑制线虫par-1 基因的表达；三年后，Fire等注入双链RNA到线虫中发现比注入单链更有效，随后发展了通过RNAi进行线虫基因敲除的策略。 2001年Elbashir等《 Nature》上首次报道通过21个核苷酸的siRNA成功地在哺乳动物培养细胞中诱导特异性基因沉默。随后，在小鼠等多种细胞中也取得了成功。2002---载体 * 化学合成 体外合成siRNA对应的DNA发卡模板 siRNA进入细胞（转染） siRNA设计 siRNA合成 体外转录合成 siRNA标记 模板构建到载体 质粒进入细胞（转染，转化） 检测（核酸，蛋白质水平） 目的基因片段的PCR扩增 PCR产物转录为RNA长片段 RNase III消化 * 体外转录合成SiRNA * U6/H1-siRNA 表达载体的构建 Tandem Type Hairpin Type GN17C TTTTT U6 P U6 P GN17C TTTTT GN18-loop-N18 U6 P TTTTT Sense antisense antisense Sense GN17CU4 4UCN17G 转录 退火 siRNA GN18 4UCN18 GN18NN 4UCN18 转录 Dicer siRNA * injected dsRNA — a mixture of both sense and antisense strands — into C. elegans (10). This injection resulted in much more efficient silencing than injection of either the sense or the antisense strands alone. Indeed, injection of just a few molecules of dsRNA per cell was sufficient to completely silence the homologous genes expression. Furthermore, injection of dsRNA into the gut of the worm caused gene silencing not only throughout the worm, but also in its first generation offspring (10). The potency of RNAi inspired Fire and Timmons to try feeding nematodes bacteria that had been engineered to express dsRNA homologous to the C. elegans unc-22 gene. Surprisingly, these worms developed an unc-22 null-like phenotype (11-13). Further work showed that soaking worms in dsRNA was also able to induce silencing (14). These strategies, whereby large numbers of nematodes are exposed to dsRNA, have enabled large-scale screens to select for RNAi-defective C. elegans mutants and have led to large numbers of gene knockout studies within this organism (15-18). Elbashir et al. (Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494.) were the first to demonstrate that gene-specific repression can be mediated by double-stra