Inhibition of Trypanosoma brucei gene expression by RNA interference usingan integratable vector with opposing T7 promoters

RNA interference is a powerful method for inhibition of gene expression in Trypanosoma brucei (Ngo, H., Tschudi, C,, Gull, K,, and Ullu, E. (1998) Pro c. Natl. Acad. Sci. U. S. A. 95, 14687-14692), Here we describe a vector (p ZJM) for in vivo tetracycline-inducible synthesis of double-stranded RNA (d sRNA) in stably transformed cells. The dsRNA is synthesized from opposing T 7 promoters. We tested the vector with genes involved in processes such as kinetoplast DNA replication, mitochondrial mRNA synthesis, glycosyl phospha tidylinositol biosynthesis, glycosome biogenesis, and polyamine biosynthesi s, In most cases the induction of dsRNA caused specific and dramatic loss o f the appropriate mRNA and in many cases there was growth inhibition or cel l death. One striking phenotype was the loss of kinetoplast DNA after inter ference with expression of a topoisomerase II. The gene being analyzed by t his procedure need not even be fully sequenced. In fact, many of the genes we tested were derived from partial sequences in the T. brucei genome data base that were identified by homology with known proteins. It takes as litt le as 3 weeks from identification of a gene sequence in the data base to th e appearance of a phenotype.