Skip to main content
×
×
Home

Genetic interference in Trypanosoma brucei by heritable and inducible double-stranded RNA

  • HUAFANG SHI (a1), APPOLINAIRE DJIKENG (a1), TOMER MARK (a1), ELIZABETH WIRTZ (a2), CHRISTIAN TSCHUDI (a1) and ELISABETTA ULLU (a1) (a3)...
    • Published online: 01 July 2000
Abstract

The use of double-stranded RNA (dsRNA) to disrupt gene expression has become a powerful method of achieving RNA interference (RNAi) in a wide variety of organisms. However, in Trypanosoma brucei this tool is restricted to transient interference, because the dsRNA is not stably maintained and its effects are diminished and eventually lost during cellular division. Here, we show that genetic interference by dsRNA can be achieved in a heritable and inducible fashion. To show this, we established stable cell lines expressing dsRNA in the form of stem-loop structures under the control of a tetracycline-inducible promoter. Targeting α-tubulin and actin mRNA resulted in potent and specific mRNA degradation as previously observed in transient interference. Surprisingly, 10-fold down regulation of actin mRNA was not fatal to trypanosomes. This type of approach could be applied to study RNAi in other organisms that are difficult to microinject or electroporate. Furthermore, to quickly probe the consequences of RNAi for a given gene we established a highly efficient in vivo T7 RNA polymerase system for expression of dsRNA. Using the α-tubulin test system we obtained greater than 98% transfection efficiency and the RNAi response lasted at least two to three cell generations. These new developments make it possible to initiate the molecular dissection of RNAi both biochemically and genetically.

Copyright
Corresponding author
Reprint requests to: Elisabetta Ullu, Department of Internal Medicine, Yale Medical School, 333 Cedar Street, New Haven, Connecticut 06520-8022, USA; e-mail: elisabetta.ullu@yale.edu.
Recommend this journal

Email your librarian or administrator to recommend adding this journal to your organisation's collection.

RNA
  • ISSN: 1355-8382
  • EISSN: 1469-9001
  • URL: /core/journals/rna
Please enter your name
Please enter a valid email address
Who would you like to send this to? *
×

Keywords

Metrics

Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed