Inhibition of Trypanosoma brucei Gene Expression by RNA Interference Using an Integratable Vector with Opposing T7 Promoters

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.

The polyadenylation complex ofTrypanosoma brucei:Characterization of the functional poly(A) polymerase

“…1,41 Cell lysates were prepared in extraction buffer [150 mM KCl, 20 mM Tris-Cl pH 7.7, 3 mM MgCl 2 , 0.5 mM DTT, containing a Complete Mini, EDTA-free protease inhibitor cocktail tablet (Roche)], and using a Dounce homogenizer, followed by sonication. Cell lysates were supplemented with 0.1% Tween-20, and centrifuged twice at 20,000 x g for 15 min to remove aggregates.…”

Identification and Functional Characterization of Lsm Proteins in Trypanosoma brucei

“…For silencing the Lsm3 gene, PCR was used to amplify the gene that was cloned into the pZJM vector carrying the two T7 opposing promoters (35). The constructs were linearized to integrate into the non-transcribed rRNA spacer region (35). One day after transfection, the cells were diluted onto the microtiter plates to obtain a clonal population.…”

“…Lines-The stem-loop construct for silencing Lsm8 was established as described in (35). Briefly, a 400-bp coding region sequence derived from the Lsm8 (GenBank TM accession number AY551266) gene obtained from the genome data base was amplified with oligonucleotides L8001 and L8003, containing the XbaI and HindIII sites, respectively, and subcloned into the NheI and HindIII site of the pJM326 vector.…”

“…The Lsm8 was silenced by producing dsRNA in the form of a stem-loop structure, as described under "Experimental Procedures" (35,37). For silencing the Lsm3 gene, PCR was used to amplify the gene that was cloned into the pZJM vector carrying the two T7 opposing promoters (35). The constructs were linearized to integrate into the non-transcribed rRNA spacer region (35).…”

“…The expression of Lsm proteins was silenced by the two approaches. The Lsm8 was silenced by producing dsRNA in the form of a stem-loop structure, as described under "Experimental Procedures" (35,37). For silencing the Lsm3 gene, PCR was used to amplify the gene that was cloned into the pZJM vector carrying the two T7 opposing promoters (35).…”