کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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2829922 | 1570657 | 2011 | 5 صفحه PDF | دانلود رایگان |
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.
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► 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.
Journal: Molecular and Biochemical Parasitology - Volume 178, Issues 1–2, July–August 2011, Pages 51–55