Abstract
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.
Highlights
Trypanosoma brucei is the protozoan parasite that causes sleeping sickness, a fatal disease that currently affects several hundred thousand people in sub-Saharan Africa
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
One advantage of RNA interference (RNAi) over conventional gene knockout approaches is that only a few hundred base pairs of a gene sequence is required. This feature is noteworthy because the T. brucei genome project is rapidly moving forward and currently there are at least partial sequences available for most of the genes of the parasite
Summary
Plasmid Constructs—Two vectors for RNAi were constructed by modifying pLew100 (a generous gift from Drs Elizabeth Wirtz and George Cross, Rockefeller University [21]). A ϳ 500-bp fragment of the gene of interest (target gene) was amplified by PCR from T. brucei 427 DNA using primers containing XbaI and HindIII linkers. This product was ligated into the NheI/HindIII sites of pJM326 (a gift from Dr Stephen Gould, Johns Hopkins University [22]). This plasmid carried the gene for an irrelevant Myc-tagged hu-. DNA polymerase ; ODC, ornithine decarboxylase; dsRNA, doublestranded RNA; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase(s)
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