Genetic interference in Trypanosoma brucei by heritable and inducible double-stranded RNA

The use of double-stranded RNA (dsRNA) to disrupt gene expression has becom e a powerful method of achieving RNA interference (RNAi) in a wide variety of organisms. However, in Trypanosoma brucei this tool is restricted to tra nsient interference, because the dsRNA is not stably maintained and its eff ects 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 expressin g dsRNA in the form of stem-loop structures under the control of a tetracyc line-inducible promoter. Targeting alpha-tubulin and actin mRNA resulted in potent and specific mRNA degradation as previously observed in transient i nterference. Surprisingly, 10-fold down regulation of actin mRNA was not fa tal to trypanosomes. This type of approach could be applied to study RNAi i n other organisms that are difficult to microinject or electroporate. Furth ermore, to quickly probe the consequences of RNAi for a given gene we estab lished a highly efficient in vivo T7 RNA polymerase system for expression o f dsRNA. Using the alpha-tubulin test system we obtained greater than 98% t ransfection efficiency and the RNAi response lasted at least two to three c ell generations. These new developments make it possible to initiate the mo lecular dissection of RNAi both biochemically and genetically.