Multiplex analysis of RNA interference defects in Trypanosoma brucei
Multiplex analysis of RNA interference defects in Trypanosoma brucei
复制标题
DOI：
10.1016/j.molbiopara.2004.11.001
复制
发表时间：
2005-01-01
影响因子：
1.5
通讯作者：
Horn, D
中科院分区：
文献类型：
--
作者：
Alsford, S;Glover, L;Horn, D
Genome sequencing is now complete for a number of trypanosomatids [1](see www. genedb. org) but functional analysis techniques that allow an increase in throughput are urgently needed if we are to fully exploit this data. RNA interference (RNAi) is a biological response to double-stranded RNA (dsRNA) that knocks down expression from complementary genes. RNAi has revolutionised research on gene function, been exploited as a high-throughput experimental tool [2] and is currently the method of choice for loss-offunction experiments in Trypanosoma brucei. We have established a simple quantitative RNAi barcode methodology that generates a read-out of relative cell number from a mixed T. brucei culture engineered for inducible RNAi. The RNAi target sequence, usually between 400 and 600 bp, is flanked by head-to-head inducible promoters and stably integrated into the genomic DNA of each cell type. Each target then serves as the template for dsRNA synthesis and as the barcode that can be amplified and labelled through multiplex competitive PCR with a single primer. The mixed barcode probe is then hybridised to specific sequences to produce the read-out. Proof-of-principle experiments demonstrate that the read-out can provide a quantitative report of relative drug resistance, relative RNAi-induced growth defects and RNAi-induced auxotrophy. Increased throughput, a range of alternative assay systems and read-out using microarray should all be compatible with the methodology.A construct with head-to-head tetracycline inducible promoters is commonly used for RNAi experiments in T. brucei [3]. We routinely select specific gene targets using the RNAit software [4](see http://trypanofan. path. cam. ac. uk/software/RNAit. html) that are then amplified by PCR, cloned in the p2T7TAblue RNAi vector (Fig. 1A), which has been optimised for direct cloning of PCR products [5], and introduced into T. brucei. Throughput can be limited by laborious phenotype analysis and subtle defects can be difficult to confirm however. We have explored the use of RNAi barcode analysis to report relative proportions of bloodstream-form cells within mixed cultures.
