@article{cc2f4f1ddb8947bf8582ce2506c53c29,
title = "A doubly inducible system for RNA interference and rapid RNAi plasmid construction in Trypanosoma brucei",
abstract = "The most rapid method for the generation of conditional mutants in Trypanosoma brucei is the use of RNA interference. A single copy of the target sequence is cloned between two opposing T7 promoters bearing tet operators, and the resulting plasmid is integrated into the genome of cells expressing both the tet repressor and T7 RNA polymerase. Upon addition of tetracycline, double-stranded RNA is synthesised from the two T7 promoters. Unfortunately, repression of T7 promoter activity may sometimes be insufficient to prevent expression of toxic amounts of double-stranded RNA. We describe here cell lines in which the expression of T7 polymerase is under tetracycline control, and show that regulation of polymerase expression can modulate transcription from a constitutive T7 promoter. In addition we describe a construct containing two copies of the tn10 Tet repressor for easy creation of repressor-expressing trypanosomes, and an RNA interference vector which allows {"}TA{"} cloning of unmodified PCR products and blue/white selection.",
keywords = "RNA interference vector, T7 polymerase, Tet repressor, Trypanosoma brucei",
author = "Alibu, {Vincent P.} and Lilian Storm and Simon Haile and Christine Clayton and David Horn",
note = "Funding Information: We thank Doug LaCount and John Donelson for the p2T7 Ti B vector, Dominique Soldati (then at the ZMBH) for a sample of antibodies to the Tet repressor, and Achim Schnaufer (Seattle Biomedical research Institute, Washington, Seattle) for anti-T7 antibodies. We also thank Mark Field and Clare Allen (Imperial College, London) for the clathrin fragment, and George Cross for useful comments. This work was supported by the Wellcome Trust; VPA is supported by the World Health Organization programme in Research and Training in Tropical Diseases (TDR). DH constructed and tested p2T7 Ti TAblue. LS built all the other new plasmids, and did all the work on the 927 lines and all the CAT transfections and assays on the bloodstream cells with inducible T7 polymerase. SH made bloodstream form 427–1313–514 and some 427–1313–1333 lines. VPA made the procyclic form 427–1313–514 and 427–1313–1333, did the Western blots and tested all cell lines for RNAi sensitivity. DH and CC supervised the work and wrote the paper with assistance from VPA and LS.",
year = "2005",
month = jan,
doi = "10.1016/j.molbiopara.2004.10.002",
language = "English",
volume = "139",
pages = "75--82",
journal = "Molecular and Biochemical Parasitology",
issn = "0166-6851",
publisher = "Elsevier",
number = "1",
}