Introduction to RNAi in Trypanosomes

In December 1998 the Ullu lab at Yale published the first report of dsRNA-mediated mRNA degradation in Trypanosoma brucei (Ngo et al., 1998). These experiments used either electroporation of in vitro synthesized dsRNA or transient in vivo expression of single-strand RNA that forms a stem–loop structure capable of inducing RNAi. At that time, the arsenal of genetic techniques available to the trypanosome researcher was limited (Clayton, 1999). For example, gene knockout was possible through homologous recombination, although the diploid genome of T. brucei complicated this approach. Furthermore, essential genes were difficult to examine by knockout, necessitating complex strategies in which inducible ectopic expression needed to be maintained while the genomic knockouts were generated (Wirtz et al., 1999). RNAi raised hopes for a powerful and convenient genetic approach for these eukaryotes.

Our lab has made extensive use of RNAi in studying gene function in T. brucei, the eukaryotic parasite that causes African sleeping sickness. The purpose of this chapter is to review the steps our lab has taken in developing an inducible RNAi system that has allowed us to achieve the goal of bona fide RNAi-based forward genetics in T. brucei. In addition, this chapter will review our development of an easy-to-use, inducible RNAi system, presenting a few examples of how this approach has allowed us to gain new insights into gene function, especially in the case of essential genes.