five ways (专外).ppt

体内表达 ??? 前面的3种方法主要都是体外制备siRNAs，并且需要专门的RNA转染试剂将siRNAs转到细胞内。而采用siRNA表达载体和基于PCR的表达框架则属于：从转染到细胞的DNA模版中在体内转录得到siRNAs。这两种方法的优点在于不需要直接操作RNA。 In Vivo ExpressionAll of the methods described so far rely on the in vitro preparation of siRNAs. The use of siRNA expression vectors and PCR-based expression cassettes, however, relies on in vivo transcription of siRNAs from DNA templates introduced into cells. A major advantage of these two approaches is that there is no need to work directly with RNA. 4. siRNA表达载体 ??? 多数的siRNA表达载体依赖3种RNA聚合酶III 启动子(pol III)中的一种，操纵一段小的发夹siRNA在哺乳动物细胞中的表达。这3类启动子包括大家熟悉的人源和鼠源的U6启动子和人H1启动子。 Method #4: siRNA Expression VectorsMost siRNA expression vectors rely on one of three different RNA polymerase III (pol III) promoters to drive the expression of a small hairpin siRNA in mammalian cells . These promoters include the well-characterized human and mouse U6 promoters and the human H1 promoter. RNA pol III was chosen to drive siRNA expression because it naturally expresses relatively large amounts of small RNAs in mammalian cells and it terminates transcription upon incorporating a string of 3–6 uridines. 之所以采用RNA pol III启动子是由于它可以在哺乳动物细胞中表达更多的小分子RNA，而且它是通过添加一串（3到6个）U来终止转录的。 To use these vectors, two oligodeoxynucleotides encoding the desired short hairpin RNA sequence are ordered, annealed, and cloned into the vector downstream of the pol III promoter. 要使用这类载体，需要订购2段编码短发夹RNA序列的DNA单链，退火，克隆到相应载体的pol III 启动子下游。 * * RNA Interference: Five Ways to Produce siRNAs RNA Interference: Five Ways to Produce siRNAs Choose the Best Method for Your Research RNAi：制备siRNAs的5种方法 如何选择最适合你的方法 the central dogma： DNA transcription translation Reverse transcription Replication RNA Protein RNAi 1．Five Ways to Produce siRNAs More and more researchers are using small interfering RNAs (siRNAs) to reduce the expression of specific mammalian genes. 越来越多的研究人员开始采用小分子干扰RNA（small interfering RNAs，siRNAs)来抑制特定的哺乳动物基因表达。 siRNAs are short, double stranded RNA molecules that