A Gateway® compatible vector for gene silencing in bloodstream form Trypanosoma brucei

Savitha Kalidas, Qiong Li, Margaret A. Phillips

Research output: Contribution to journalArticlepeer-review

19 Scopus citations

Abstract

RNA interference is the most rapid method for generation of conditional knockdown mutants in Trypanosoma brucei. The dual T7 promoter (pZJM) and the stem-loop vectors have been widely used to generate stable inducible RNAi cell lines with the latter providing tighter regulatory control. However, the steps for cloning stem-loop constructs are cumbersome requiring either multiple cloning steps or multi-fragment ligation reactions. We report the development of a vector (pTrypRNAiGate) derived from pLEW100 that utilizes the Gateway ® recombination system to facilitate easy production of hairpin RNA constructs. This approach allows the final stem-loop RNAi construct to be generated from a single cloning step of the PCR-derived gene fragment followed by an in vitro recombination reaction. The new vector facilitates high-throughput applications for gene silencing and provides a tool for functional genomics in T. brucei.

Original languageEnglish (US)
Pages (from-to)51-55
Number of pages5
JournalMolecular and Biochemical Parasitology
Volume178
Issue number1-2
DOIs
StatePublished - Jul 1 2011

Keywords

  • Gateway vector
  • RNAi
  • T. brucei
  • pTrypRNAiGate

ASJC Scopus subject areas

  • Parasitology
  • Molecular Biology

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