期刊
MOLECULAR AND BIOCHEMICAL PARASITOLOGY
卷 178, 期 1-2, 页码 51-55出版社
ELSEVIER
DOI: 10.1016/j.molbiopara.2011.03.002
关键词
RNAi; Gateway vector; pTrypRNAiGate; T. brucei
资金
- National Institutes of Health [R01 AI34432, R01 AI078962]
- Welch Foundation [I-1257]
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. (C) 2011 Elsevier B.V. All rights reserved.
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