Down-Regulating Gene Expression by RNA Interference in Trypanosoma brucei

RNA interference (RNAi) in Trypanosoma brucei was first reported in 1998 (1 ). As in other eukaryotes, interference involves digestion of the interfering double-stranded RNA into short fragments (2 ), a polysome-associated complex (3 ), and Argonaute protein (4 ,5 ). T. brucei is an ideal organism for testing gene function by RNAi. A complete genome sequence is available, and liquid suspension culture is unproblematic. Various combinations of RNAi vector and host trypanosomes, with different advantages, are available; if procedures are working optimally, it should be possible to obtain a stable cell line with inducible RNAi within 2 wk. The RNAi process itself is apparently not essential for parasite survival (4 ), although some adverse effects have been reported (5 ). To analyze the process itself, it is possible to delete candidate genes completely, as the efficiency of homologous recombination is essentially 100%; in situ epitope tagging can also be effected through homologous recombination (6 ). For general reviews of the peculiarities of trypanosome gene expression and RNA processing, see refs. 7 and 8 , and for pre-RNAi methods, such as inducible gene expression and knockouts by homologous recombination, see ref. 9 . A few recent applications of RNAi in trypanosomes are found in refs. 10 –16 . Importantly, in trypanosomes, RNAi can also be used to deplete nuclear RNAs (8 ,17 ). In this chapter, we will describe two different options for the construction of RNAi vectors, followed by methods for trypanosome culture and selection of transfectants. At the end, we will highlight various options for testing of phenotypes, as well as various pitfalls with working with RNAi in trypanosomes.