Shi H, Djikeng A, Mark T, Wirtz E, Tschudi C, Ullu E
Department of Internal Medicine, Yale Medical School, New Haven, Connecticut 06520-8022, USA.
RNA. 2000 Jul;6(7):1069-76. doi: 10.1017/s1355838200000297.
The use of double-stranded RNA (dsRNA) to disrupt gene expression has become a powerful method of achieving RNA interference (RNAi) in a wide variety of organisms. However, in Trypanosoma brucei this tool is restricted to transient interference, because the dsRNA is not stably maintained and its effects are diminished and eventually lost during cellular division. Here, we show that genetic interference by dsRNA can be achieved in a heritable and inducible fashion. To show this, we established stable cell lines expressing dsRNA in the form of stem-loop structures under the control of a tetracycline-inducible promoter. Targeting a-tubulin and actin mRNA resulted in potent and specific mRNA degradation as previously observed in transient interference. Surprisingly, 10-fold down regulation of actin mRNA was not fatal to trypanosomes. This type of approach could be applied to study RNAi in other organisms that are difficult to microinject or electroporate. Furthermore, to quickly probe the consequences of RNAi for a given gene we established a highly efficient in vivo T7 RNA polymerase system for expression of dsRNA. Using the alpha-tubulin test system we obtained greater than 98% transfection efficiency and the RNAi response lasted at least two to three cell generations. These new developments make it possible to initiate the molecular dissection of RNAi both biochemically and genetically.
利用双链RNA（dsRNA）干扰基因表达已成为在多种生物体中实现RNA干扰（RNAi）的一种强大方法。然而，在布氏锥虫中，这种工具仅限于瞬时干扰，因为dsRNA不能稳定维持，其效应在细胞分裂过程中会减弱并最终消失。在此，我们表明dsRNA介导的遗传干扰能够以可遗传且可诱导的方式实现。为证明这一点，我们构建了稳定细胞系，这些细胞系在四环素诱导型启动子的控制下，以茎环结构形式表达dsRNA。靶向α-微管蛋白和肌动蛋白mRNA导致了有效的、特异性的mRNA降解，这与之前在瞬时干扰中观察到的情况一致。令人惊讶的是，肌动蛋白mRNA下调10倍对锥虫并不致命。这种方法可应用于研究其他难以进行显微注射或电穿孔的生物体中的RNAi。此外，为了快速探究RNAi对特定基因的影响，我们建立了一种高效的体内T7 RNA聚合酶系统用于dsRNA的表达。使用α-微管蛋白测试系统，我们获得了大于98%的转染效率，并且RNAi反应至少持续两到三个细胞世代。这些新进展使得从生化和遗传角度对RNAi进行分子剖析成为可能。
