Single-locus targeting constructs for reliable regulated RNAi and transgene expression in Trypanosoma brucei

被引：108

作者：

Alsford, Sam ^{[1
]}

Horn, David ^{[1
]}

机构：

[1] Univ London London Sch Hyg & Trop Med, London WC1E 7HT, England

来源：

MOLECULAR AND BIOCHEMICAL PARASITOLOGY | 2008年 / 161卷 / 01期

基金：

英国惠康基金;

关键词：

epigenetic; position-effects; RNAi; localisation; functional-genomics;

D O I：

10.1016/j.molbiopara.2008.05.006

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

A major obstacle to reproducible expression of recombinant transcripts lies in the epigenetic effects of the flanking chromatin following integration. We previously presented a strategy to overcome this problem in bloodstream form Trypanosoma brucei, using a reporter to identify a ribosomal-spacer locus that supports optimal expression and then marking that locus for subsequent targeting. Advantages include elimination of variable-expression position-effects and the easy confirmation of correct integration. We now report a set of validated constructs that exploit this system for expression of dsRNA or recombinant protein. The current construct-set allows expression of intramolecular dsRNA for RNA interference knockdown or expression of proteins that can incorporate c-Myc epitope(s) or a fluorescent-tag for subcellular localisation, interaction and/or other functional analysis. The constructs are integrated at a single, marked locus and deliver reliable and reproducible expression. (C) 2008 Elsevier B.V. All rights reserved.

引用

页码：76 / 79

页数：4

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