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

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.

Graphical abstractFigure optionsDownload full-size imageDownload high-quality image (104 K)Download as PowerPoint slideHighlights► Development of a Gateway vector for generation of stem loop RNAi constructs for Trypanosoma brucei. ► Using this approach the final vector is generated via cloning of a single PCR fragment followed by a recombination reaction. ► New vector tested for three polyamine biosynthetic genes and shown to give similar results to standard stem-loop constructs. ► Vector facilitates rapid cloning of stem loop RNAi constructs.