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. 2004 Oct;14(10B):2162-8.
doi: 10.1101/gr.2505604.

Toward improving Caenorhabditis elegans phenome mapping with an ORFeome-based RNAi library

Jean-François Rual  1 , Julian Ceron, John Koreth, Tong Hao, Anne-Sophie Nicot, Tomoko Hirozane-Kishikawa, Jean Vandenhaute, Stuart H Orkin, David E Hill, Sander van den Heuvel, Marc Vidal
Affiliations

Toward improving Caenorhabditis elegans phenome mapping with an ORFeome-based RNAi library

Jean-François Rual et al. Genome Res. 2004 Oct.

Abstract

The recently completed Caenorhabditis elegans genome sequence allows application of high-throughput (HT) approaches for phenotypic analyses using RNA interference (RNAi). As large phenotypic data sets become available, "phenoclustering" strategies can be used to begin understanding the complex molecular networks involved in development and other biological processes. The current HT-RNAi resources represent a great asset for phenotypic profiling but are limited by lack of flexibility. For instance, existing resources do not take advantage of the latest improvements in RNAi technology, such as inducible hairpin RNAi. Here we show that a C. elegans ORFeome resource, generated with the Gateway cloning system, can be used as a starting point to generate alternative HT-RNAi resources with enhanced flexibility. The versatility inherent to the Gateway system suggests that additional HT-RNAi libraries can now be readily generated to perform gene knockdowns under various conditions, increasing the possibilities for phenome mapping in C. elegans.

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Figures

Figure 1
Figure 1
Generating RNAi resources from flexible Gateway ORFeome and promoterome collections. AntR1 and AntR2 refer to antibiotic resistance markers number 1 and 2.
Figure 2
Figure 2
Overview of the RNAi screening procedure. (A) Overnight cultures of 96 bacterial clones are inoculated in sixteen 24-wells plates (six clones per plate and four wells per clone). After overnight induction of the dsRNA synthesis, three to 10 worms synchronized at the L1-stage (N2 or lin-35 strain) were deposited into the wells. (B) RNAi experiments were performed at 20°C. We observed a wide range of phenotypes across development.
See this image and copyright information in PMC

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WEB SITE REFERENCES

    1. http://www.wormbase.org; Source for C. elegans genome and RNAi data.
    1. http://worfdb.dfci.harvard.edu; Source for C. elegans ORFeome data.
    1. http://www.openbiosystems.com; Source for request of ORFeome-RNAi v1.1 clones.
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