Inducible RNAi as a forward genetic tool in <span class='italic'>Trypanosoma brucei</span>

doi:10.1017/CBO9780511546402.021

18 - Inducible RNAi as a forward genetic tool in Trypanosoma brucei

Published online by Cambridge University Press: 31 July 2009

Zefeng Wang and

Edited by

Foreword by

Andrew Fire and

Marshall Nirenberg

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Mark E. Drew: Affiliation:
Dept. of Mol. Microbiology, Washington University School of Medicine
Shawn A. Motyka: Affiliation:
Department of Biological Chemistry, Johns Hopkins Medical School
James C. Morris: Affiliation:
Department of Genetics, Biochemistry and Life Science Studies, Clemson University
Zefeng Wang: Affiliation:
Dept. of Mol. Microbiology, Washington University School of Medicine
Paul T. Englund: Affiliation:
Department of Biological Chemistry, Johns Hopkins Medical School
Krishnarao Appasani: Affiliation:
GeneExpression Systems, Inc., Massachusetts
Andrew Fire: Affiliation:
Stanford University, California

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Summary

Introduction to RNAi in Trypanosomes

In December 1998 the Ullu lab at Yale published the first report of dsRNA-mediated mRNA degradation in Trypanosoma brucei (Ngo et al., 1998). These experiments used either electroporation of in vitro synthesized dsRNA or transient in vivo expression of single-strand RNA that forms a stem–loop structure capable of inducing RNAi. At that time, the arsenal of genetic techniques available to the trypanosome researcher was limited (Clayton, 1999). For example, gene knockout was possible through homologous recombination, although the diploid genome of T. brucei complicated this approach. Furthermore, essential genes were difficult to examine by knockout, necessitating complex strategies in which inducible ectopic expression needed to be maintained while the genomic knockouts were generated (Wirtz et al., 1999). RNAi raised hopes for a powerful and convenient genetic approach for these eukaryotes.

Our lab has made extensive use of RNAi in studying gene function in T. brucei, the eukaryotic parasite that causes African sleeping sickness. The purpose of this chapter is to review the steps our lab has taken in developing an inducible RNAi system that has allowed us to achieve the goal of bona fide RNAi-based forward genetics in T. brucei. In addition, this chapter will review our development of an easy-to-use, inducible RNAi system, presenting a few examples of how this approach has allowed us to gain new insights into gene function, especially in the case of essential genes.

Type: Chapter
Information: RNA Interference Technology
From Basic Science to Drug Development
, pp. 247 - 256

DOI: https://doi.org/10.1017/CBO9780511546402.021 [Opens in a new window]

Publisher: Cambridge University Press

Print publication year: 2005

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