Genome-wide RNAi screens in bloodstream form trypanosomes identify drug transporters

An improved transfection procedure was used to generate an RNAi library in bloodstream form Trypanosoma brucei. A genome-wide screen identified an amino acid transporter mediating sensitivity to eflornithine. [Display omitted] ▶ Construction of an RNAi library with 9-fold genome coverage in bloodstream form T. brucei. ▶ Improved transfection protocol using a simple buffer of defined composition. ▶ Unbiased approach to gene discovery. ▶ Identification of an amino acid transporter mediating sensitivity to eflornithine. An inducible RNA interference (RNAi) library, consisting of a pool of independent stable transformants with 9-fold genome coverage, was constructed in bloodstream form Trypanosoma brucei using an improved transfection protocol. RNAi induction and selection of resistant parasites was performed in the presence of melarsoprol or eflornithine. The former led to the isolation of the adenosine transporter TbAT1, which is known to be involved in melarsoprol uptake, while the latter identified an amino acid transporter, AAT6. Knockdown of AAT6 reduced mRNA levels to 30–35% in independent clones and increased resistance to eflornithine >5-fold. Genome-wide screens with this library allow an unbiased approach to gene discovery, are extremely rapid and do not exclude essential genes.