In 1998, C. elegans was the first multicellular eukaryote to have its genome completely sequenced, revealing 97 Mb of sequence encoding approximately 19,000 predicted genes (The C. elegans sequencing consortium, 1998). Also in 1998, RNA interference (RNAi) was discovered (Fire et al, 1998): the introduction of double-stranded RNA results in potent and specific inactivation of an endogenous gene with corresponding sequence. The RNAi technique enables rapid, targeted gene inactivation, and has become an extremely important tool for studying gene function in vivo. RNAi can be performed in C. elegans by injecting dsRNA (Fire et al, 1998), by soaking worms in a solution of dsRNA (Tabara et al, 1998), or by feeding them Escherichia coli expressing target-gene dsRNA (Timmons and Fire, 1998). We constructed an RNAi feeding library of 16,757 bacterial strains (targeting ~86% of predicted genes) for use in genome-wide RNAi screening in C. elegans (Fraser et al, 2000; Kamath et al, 2003). Although RNAi is a reverse genetic tool, when used on a large scale it is essentially a forward genetic screening method. The library can be used to screen for genes involved in nearly any process of interest, to identify the majority of genes acting that process, with no necessary downstream cloning. For example, we conduct screens for genes involved in cell polarity and chromatin regulation. In addition, having functional data covering most of the genome allows large-scale analyses such as genomic clustering and evolutionary studies to be performed.
RNAi Library
Individual clones or the entire RNAi library described in Fraser et al, 2000 and Kamath et al, 2003 can be ordered directly from Geneservice, Ltd. We are currently preparing ~4000 new RNAi bacterial strains to complete the library. These will be distributed by Geneservice when finished.
Clone Verification
In making the RNAi library, several quality control steps were carried out to reduce the risk of library clones with the incorrect insert. Despite these precautions, clone errors do occur at low frequency in the library (current estimate is 7%).
The Sanger Centre kindly carried out a round of sequencing of our RNAi library, yielding information for 87% of RNAi bacterial strains.
A list of correct clones can be found here.
A list of incorrect clones can be found here, but note that there are false positives on this list (i.e., some clones are actually correct). Upon re-sequencng a subset, 30% were found to have the correct insert.
Clones not on either list have no sequencing information.
After conducting RNAi screens with the feeding library, positive clones should still be sequenced to verify the identity of the insert.
Methods
Our current lab RNAi feeding protocol.
Wormbook chapter on RNAi.
Protocol for preparing TA cloning vector.
Chapter on designing RNAi screens.