title: A Gateway® compatible vector for gene silencing in bloodstream form Trypanosoma brucei.
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.
authors.affiliation: Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390-9041, United States.
authors.forename: Savitha
authors.initials: S
authors.lastname: Kalidas
authors.forename: Qiong
authors.initials: Q
authors.lastname: Li
authors.forename: Margaret A
authors.initials: MA
authors.lastname: Phillips
mesh_terms: D003001:Cloning, Molecular / Q000379:methods; D020868:Gene Silencing; D005822:Genetic Vectors; D005827:Genetics, Microbial / Q000379:methods*; D017902:Podoviridae / Q000235:genetics; D034622:RNA Interference; D014346:Trypanosoma brucei brucei / Q000235:genetics*