Functional genomic analysis of C. elegans chromosome I by systematic RNA interference

Abstract

Complete genomic sequence is known for two multicellular eukaryotes, the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster, and it will soon be known for humans. However, biological function has been assigned to only a small proportion of the predicted genes in any animal. Here we have used RNA-mediated interference (RNAi) to target nearly 90% of predicted genes on C. elegans chromosome I by feeding worms with bacteria that express double-stranded RNA. We have assigned function to 13.9% of the genes analysed, increasing the number of sequenced genes with known phenotypes on chromosome I from 70 to 378. Although most genes with sterile or embryonic lethal RNAi phenotypes are involved in basal cell metabolism, many genes giving post-embryonic phenotypes have conserved sequences but unknown function. In addition, conserved genes are significantly more likely to have an RNAi phenotype than are genes with no conservation. We have constructed a reusable library of bacterial clones that will permit unlimited RNAi screens in the future; this should help develop a more complete view of the relationships between the genome, gene function and the environment.

p53-dependent inhibition of mammalian cell survival by a genetically selected peptide aptamer that targets the regulatory subunit of protein kinase CK2

“…These data again support of the notion that at least some aspects of CK2b functions are not based on its interaction with the CK2 catalytic subunit (Filhol et al, 2004;Bibby and Litchfield, 2005). In mice as well as in Caenorhabditis elegans, a functional loss of CK2b is lethal, demonstrating that the protein is essential for viability (Fraser et al, 2000;Buchou et al, 2003). These genetic knockouts also illustrate the dramatic phenotypic consequences of disrupting all the potential interactions in which the CK2b protein is involved.…”

A zebrafish vasa morphant abolishes Vasa protein but does not affect the establishment of the germline

“…To interfere with Vasa protein levels in the early zebrafish embryo, we applied RNAi (Wargelius et al, 1999), which has been successfully applied in different organisms (Fraser et al, 2000;Misquitta and Paterson, 1999). We injected vasa dsRNA into one-or two-cellstage embryos that were raised to 24 hpf (hours postfertilization) and then were evaluated for the presence of vasa mRNA or Vasa protein by whole-mount in situ hybridization and whole-mount immunostaining, respectively.…”

The endocytic pathway mediates cell entry of dsRNA to induce RNAi silencing

“…Feeding of RNAi bacteria to worms was carried out as previously described 42,43 . Briefly, young adult worms were transferred to candidate RNAi bacteria in two different concentrations of IPTG.…”

“…HT115 bacteria transformed with RNAi vectors expressing dsRNA of the genes of interest were grown at 37 °C in LB with 10 μg mL −1 tetracycline and 50 μg mL −1 carbenicillin, then seeded onto nematode growth mediacarbenicillin plates and supplemented with two different concentrations of IPTG (0.2 and 2 μM). The presence of the insert of each clone of interest from the RNAi library was verified by PCR analysis with T7 primer.Feeding of RNAi bacteria to worms was carried out as previously described 42,43 . Briefly, young adult worms were transferred to candidate RNAi bacteria in two different concentrations of IPTG.…”