在血流形式锥虫中进行的全基因组RNA干扰筛选鉴定出药物转运蛋白。

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

作者信息

Schumann Burkard Gabriela, Jutzi Pascal, Roditi Isabel

机构信息

Institute of Cell Biology, University of Bern, Baltzerstrasse 4, CH-3012 Bern, Switzerland.

出版信息

Mol Biochem Parasitol. 2011 Jan;175(1):91-4. doi: 10.1016/j.molbiopara.2010.09.002. Epub 2010 Sep 22.

DOI:10.1016/j.molbiopara.2010.09.002

PMID:20851719

Abstract

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.

摘要

利用一种改进的转染方案，在布氏锥虫的血流形式中构建了一个诱导型RNA干扰（RNAi）文库，该文库由一组具有9倍基因组覆盖率的独立稳定转化体组成。在美拉胂醇或依氟鸟氨酸存在的情况下进行RNAi诱导和抗性寄生虫的筛选。前者导致了腺苷转运体TbAT1的分离，已知该转运体参与美拉胂醇的摄取，而后者鉴定出一种氨基酸转运体AAT6。在独立克隆中，AAT6的敲低使mRNA水平降至30%-35%，并使对依氟鸟氨酸的抗性增加了5倍以上。用该文库进行全基因组筛选可采用无偏向性方法进行基因发现，速度极快且不排除必需基因。