Analysis of Gene Function in Trypanosoma brucei Using RNA Interference

Part of the Methods in Molecular Biology book series (MIMB, volume 265)


Trypanosoma brucei, a flagellate protozoa of the family Trypanosomatidae, has become one of the model systems for unicellular pathogens to study fundamentally important biological phenomena. Currently, the method of choice to examine gene function in these organisms is RNA interference (RNAi). mRNA degradation is triggered by double-stranded RNA (dsRNA) produced in vivo from transgenes transcribed from opposing tetracycline (tet)-inducible T7 RNA polymerase promoters, or hairpin RNA transcribed from the tet-inducible procyclic acidic repetitive protein promoter. In this chapter, we describe some of the methods we employ for ablation of gene expression by RNAi in T. brucei with particular emphasis on transfection and cloning of procyclic cells, induction of dsRNA expression, isolation of RNA and analysis of dsRNA, and target mRNA.

Key Words

DNA transfection hairpin construct Trypanosoma brucei clonal cell line RNA dot blot inducible promoter 


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Copyright information

© Humana Press Inc. 2004

Authors and Affiliations

  1. 1.Department of Internal MedicineYale University School of MedicineNew Haven
  2. 2.Department of Epidemiology and Public HealthYale University School of MedicineNew Haven
  3. 3.Department of Cell BiologyYale University School of MedicineNew HavenCT

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