LaCount D J, Bruse S, Hill K L, Donelson J E
Department of Biochemistry, College of Medicine, 4-403 Bowen Science Building, 51 Newton Road, University of Iowa, Iowa City, IA 52242, USA.
Mol Biochem Parasitol. 2000 Nov;111(1):67-76. doi: 10.1016/s0166-6851(00)00300-5.
The discovery of double-stranded RNA interference (dsRNAi) in Trypanosoma brucei provides a convenient method to generate knockout phenotypes in this protozoan parasite [Ngo H, Tschudi C, Gull K, Ullu E. Double-stranded RNA induces mRNA degradation in Trypanosoma brucei. Proc Natl Acad Sci USA 1998;95:14687-14692]. The presence of double-stranded RNA (dsRNA) dominantly silences gene expression in a sequence-specific manner by causing the corresponding endogenous RNA to be degraded. To simplify the generation of knockout phenotypes in T. brucei via dsRNAi, we used two promoters arranged as an inverted repeat on a plasmid. This promoter arrangement generates transcripts of both strands of DNA inserted between the promoters, which then form dsRNA. We have used plasmids encoding either two T. brucei ribosomal RNA promoters or two bacteriophage T7 promoters to interfere with expression of alpha-tubulin (TUB), green fluorescent protein (GFP), paraflagellar rod protein A (PFRA), flagellum-adhesion glycoprotein 1 (FLA1), and histone 2B (H2B) in T. brucei. We show here that FLA1 is required for flagellar attachment in T. brucei and that H2B is required for parasite growth. Thus, the two-promoter approach efficiently generates dsRNAi in T. brucei and can be used to produce both specific and random knockout phenotypes in T. brucei. This approach should be useful in generating knockout phenotypes in other kinetoplastid parasites including Trypanosoma cruzi and Leishmania.
在布氏锥虫中发现双链RNA干扰（dsRNAi）为在这种原生动物寄生虫中产生基因敲除表型提供了一种便捷方法[Ngo H, Tschudi C, Gull K, Ullu E. 双链RNA诱导布氏锥虫mRNA降解. 美国国家科学院院刊1998;95:14687 - 14692]。双链RNA（dsRNA）的存在通过导致相应的内源性RNA降解，以序列特异性方式显著沉默基因表达。为了通过dsRNAi简化布氏锥虫中基因敲除表型的产生，我们在质粒上使用了两个反向排列的启动子。这种启动子排列产生插入在启动子之间的DNA两条链的转录本，然后形成dsRNA。我们使用编码两个布氏锥虫核糖体RNA启动子或两个噬菌体T7启动子的质粒来干扰布氏锥虫中α - 微管蛋白（TUB）、绿色荧光蛋白（GFP）、副鞭毛杆蛋白A（PFRA）、鞭毛粘附糖蛋白1（FLA1）和组蛋白2B（H2B）的表达。我们在此表明，FLA1是布氏锥虫鞭毛附着所必需的，而H2B是寄生虫生长所必需的。因此，双启动子方法在布氏锥虫中能有效地产生dsRNAi，可用于在布氏锥虫中产生特异性和随机的基因敲除表型。这种方法在包括克氏锥虫和利什曼原虫在内的其他动基体寄生虫中产生基因敲除表型时应该是有用的。
