Genome-wide RNAi screens in bloodstream form trypanosomes identify drug transporters.
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.
Schumann Burkard
Gabriela
G
Institute of Cell Biology, University of Bern, Baltzerstrasse 4, CH-3012 Bern, Switzerland.
Jutzi
Pascal
P
Roditi
Isabel
I
D000981:Antiprotozoal Agents / Q000494:pharmacology; D001769:Blood / Q000469:parasitology*; D000518:Eflornithine / Q000494:pharmacology; D055785:Gene Knockdown Techniques / Q000379:methods*; D020868:Gene Silencing; D008549:Melarsoprol / Q000494:pharmacology; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection / Q000379:methods; D014346:Trypanosoma brucei brucei / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism
D000981:Antiprotozoal Agents; D026901:Membrane Transport Proteins; D015800:Protozoan Proteins; D008549:Melarsoprol; D000518:Eflornithine