Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 275, Issue 51, Pages 40174-40179Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M008405200
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- NIAID NIH HHS [AI21334] Funding Source: Medline
- NIGMS NIH HHS [GM27608] Funding Source: Medline
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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.
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