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

共 20 条

[1] A doubly inducible system for RNA interference and rapid RNAi plasmid construction in Trypanosoma brucei [J].

Alibu, VP ;

Storm, L ;

Haile, S ;

Clayton, C ;

Horn, D .

MOLECULAR AND BIOCHEMICAL PARASITOLOGY, 2005, 139 (01) :75-82

[2] Tagging a T-brucei RRNA locus improves stable transfection efficiency and circumvents inducible expression position effects [J].

Alsford, S ;

Kawahara, T ;

Glover, L ;

Horn, D .

MOLECULAR AND BIOCHEMICAL PARASITOLOGY, 2005, 144 (02) :142-148

[3] RNA polymerase I transcription stimulates homologous recombination in Trypanosoma brucei [J].

Alsford, Sam ;

Horn, David .

MOLECULAR AND BIOCHEMICAL PARASITOLOGY, 2007, 153 (01) :77-79

[4] The genome of the African trypanosome Trypanosoma brucei [J].

Berriman, M ;

Ghedin, E ;

Hertz-Fowler, C ;

Blandin, G ;

Renauld, H ;

Bartholomeu, DC ;

Lennard, NJ ;

Caler, E ;

Hamlin, NE ;

Haas, B ;

Böhme, W ;

Hannick, L ;

Aslett, MA ;

Shallom, J ;

Marcello, L ;

Hou, LH ;

Wickstead, B ;

Alsmark, UCM ;

Arrowsmith, C ;

Atkin, RJ ;

Barron, AJ ;

Bringaud, F ;

Brooks, K ;

Carrington, M ;

Cherevach, I ;

Chillingworth, TJ ;

Churcher, C ;

Clark, LN ;

Corton, CH ;

Cronin, A ;

Davies, RM ;

Doggett, J ;

Djikeng, A ;

Feldblyum, T ;

Field, MC ;

Fraser, A ;

Goodhead, I ;

Hance, Z ;

Harper, D ;

Harris, BR ;

Hauser, H ;

Hostetter, J ;

Ivens, A ;

Jagels, K ;

Johnson, D ;

Johnson, J ;

Jones, K ;

Kerhornou, AX ;

Koo, H ;

Larke, N .

SCIENCE, 2005, 309 (5733) :416-422

[5] RNA polymerase transcription machinery in trypanosomes [J].

Das, Anish ;

Banday, Mahrukh ;

Bellofatto, Vivian .

EUKARYOTIC CELL, 2008, 7 (03) :429-434

[6] Efficiency and specificity of RNA interference generated by intra- and intermolecular double stranded RNA in Trypanosoma brucei [J].

Durand-Dubief, M ;

Kohl, L ;

Bastin, P .

MOLECULAR AND BIOCHEMICAL PARASITOLOGY, 2003, 129 (01) :11-21

[7] Deletion of a trypanosome telomere leads to loss of silencing and progressive loss of terminal DNA in the absence of cell cycle arrest [J].

Glover, Lucy ;

Alsford, Sam ;

Beattie, Caroline ;

Horn, David .

NUCLEIC ACIDS RESEARCH, 2007, 35 (03) :872-880

[8] Codon usage suggests that translational selection has a major impact on protein expression in trypanosomatids [J].

Horn, David .

BMC GENOMICS, 2008, 9 (1)

[9] Functional genomics in Trypanosoma brucei:: A collection of vectors for the expression of tagged proteins from endogenous and ectopic gene loci [J].

Kelly, Steven ;

Reed, Jenny ;

Kramer, Susanne ;

Ellis, Louise ;

Webb, Helena ;

Sunter, Jack ;

Salje, Jeanne ;

Marinsek, Nina ;

Gull, Keith ;

Wickstead, Bill ;

Carrington, Mark .

MOLECULAR AND BIOCHEMICAL PARASITOLOGY, 2007, 154 (01) :103-109

[10] Double-stranded RNA interference in Trypanosoma brucei using head-to-head promoters [J].

LaCount, DJ ;

Bruse, S ;

Hill, KL ;

Donelson, JE .

MOLECULAR AND BIOCHEMICAL PARASITOLOGY, 2000, 111 (01) :67-76

← 1 2 →