A Gateway (R) compatible vector for gene silencing in bloodstream form Trypanosoma brucei

作者：Kalidas Savitha; Li Qiong; Phillips Margaret A^*

来源：Molecular and Biochemical Parasitology, 2011, 178(1-2): 51-55.

DOI：10.1016/j.molbiopara.2011.03.002

• RNAi
• Gateway vector
• pTrypRNAiGate
• T. brucei

RNA interference is the most rapid method for generation of conditional knockdown mutants in Trypanosoma brucei. The dual 17 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 (R) 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.

• 出版日期2011-8