Inhibition of Trypanosoma brucei gene expression by RNA interference using an 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) Proc. Natl. Acad. Sci. U. S. A. 95, 14687-14692). Here we describe a vector (pZJM) for in vivo tetracycline-inducible synthesis of double-stranded RNA (dsRNA) in stably transformed cells. The dsRNA is synthesized from opposing T7 promoters. We tested the vector with genes involved in processes such as kinetoplast DNA replication, mitochondrial mRNA synthesis, glycosyl phosphatidylinositol biosynthesis, glycosome biogenesis, and polyamine biosynthesis. In most cases the induction of dsRNA caused specific and dramatic loss of the appropriate mRNA, and in many cases there was growth inhibition or cell death. One striking phenotype was the loss of kinetoplast DNA after interference with expression of a topoisomerase II. The gene being analyzed by this 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 little as 3 weeks from identification of a gene sequence in the data base to the appearance of a phenotype.
Wang
Z
Z
Department of Biological Chemistry, Johns Hopkins School of Medicine, Baltimore, Maryland 21205, USA.
Morris
J C
JC
Drew
M E
ME
Englund
P T
PT
D000818:Animals; D017123:Bacteriophage T7 / Q000235:genetics*; D001483:Base Sequence; D017931:DNA Primers; D005786:Gene Expression Regulation / Q000235:genetics*; D017125:Genes, Protozoan; D005822:Genetic Vectors; D011401:Promoter Regions, Genetic; D016053:RNA, Protozoan / Q000037:antagonists & inhibitors*; D014346:Trypanosoma brucei brucei / Q000235:genetics*
D017931:DNA Primers; D016053:RNA, Protozoan