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BMC Research Notes

, 2:46

First Online: 23 March 2009Received: 23 February 2009Accepted: 23 March 2009DOI: 10.1186-1756-0500-2-46

Cite this article as: Monnerat, S., Clucas, C., Brown, E. et al. BMC Res Notes 2009 2: 46. doi:10.1186-1756-0500-2-46

Abstract

BackgroundThe protozoan parasite, Trypanosoma brucei, is spread by the tsetse fly and causes Human African Trypanosomiasis. Its cell cycle is complex and not fully understood at the molecular level. The T. brucei genome contains over 6000 protein coding genes with >50% having no predicted function. A small scale RNA interference RNAi screen was carried out in Trypanosoma brucei to evaluate the prospects for identifying novel cycle regulators.

ResultsProcyclic form T. brucei were transfected with a genomic RNAi library and 204 clones isolated. However, only 76 RNAi clones were found to target a protein coding gene of potential interest. These clones were screened for defects in proliferation and cell cycle progression following RNAi induction. Sixteen clones exhibited proliferation defects upon RNAi induction, with eight clones displaying potential cell cycle defects. To confirm the phenotypes, new RNAi cell lines were generated and characterised for five genes targeted in these clones. While we confirmed that the targeted genes are essential for proliferation, we were unable to unambiguously classify them as cell cycle regulators.

ConclusionOur study identified genes essential for proliferation, but did not, as hoped, identify novel cell cycle regulators. Screening of the RNAi library for essential genes was extremely labour-intensive, which was compounded by the suboptimal quality of the library. For such a screening method to be viable for a large scale or genome wide screen, a new, significantly improved RNAi library will be required, and automated phenotyping approaches will need to be incorporated.

AbbreviationsRNAiRNA interference

Nnucleus

Kkinetoplast

ORFopen reading frame

PP1protein phosphatase 1

PFprocyclic form

BSbloodstream stage.

Electronic supplementary materialThe online version of this article doi:10.1186-1756-0500-2-46 contains supplementary material, which is available to authorized users.

Séverine Monnerat, Caroline Clucas contributed equally to this work.

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Autor: Séverine Monnerat - Caroline Clucas - Elaine Brown - Jeremy C Mottram - Tansy C Hammarton

Fuente: https://link.springer.com/







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