A doubly inducible system for RNA interference and rapid RNAi plasmid construction in Trypanosoma brucei

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.