Toward Improving Caenorhabditis elegans Phenome Mapping With an ORFeome-Based RNAi Library

Center for Cancer Systems Biology and Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.
Genome Research (Impact Factor: 14.63). 11/2004; 14(10B):2162-8. DOI: 10.1101/gr.2505604
Source: PubMed


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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Available from: Tomoko Hirozane-Kishikawa, Aug 25, 2014
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    • "Plasmids containing TF ORFs were purified and transformed into E. coli HT115 bacteria, grown in Luria broth (LB) + 15 mg/ml tetracycline + 50 mg/ml ampicillin, and frozen in 5% glycerol in 96-well plates. 71 clones were obtained from the Ahringer RNAi library (Kamath et al., 2003), and five were obtained from the ORFeome RNAi library (Rual et al., 2004). GFP-or mCherry-containing clones (positive controls) and empty L4440 vector (negative control) were included in each plate for screening. "
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