Elsevier

Gene

Volume 263, Issues 1–2, 24 January 2001, Pages 103-112
Gene

Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans

https://doi.org/10.1016/S0378-1119(00)00579-5Get rights and content

Abstract

Genetic interference mediated by double-stranded RNA (RNAi) has been a valuable tool in the analysis of gene function in Caenorhabditis elegans. Here we report an efficient induction of RNAi using bacteria to deliver double-stranded RNA. This method makes use of bacteria that are deficient in RNaseIII, an enzyme that normally degrades a majority of dsRNAs in the bacterial cell. Bacteria deficient for RNaseIII were engineered to produce high quantities of specific dsRNA segments. When fed to C. elegans, such engineered bacteria were found to produce populations of RNAi-affected animals with phenotypes that were comparable in expressivity to the corresponding loss-of-function mutants. We found the method to be most effective in inducing RNAi for non-neuronal tissue of late larval and adult hermaphrodites, with decreased effectiveness in the nervous system, in early larval stages, and in males. Bacteria-induced RNAi phenotypes could be maintained over the course of several generations with continuous feeding, allowing for convenient assessments of the biological consequences of specific genetic interference and of continuous exposure to dsRNAs.

Introduction

A variety of species exhibit a defense response in which a double stranded RNA (dsRNA) ‘trigger’ produces a premature loss of endogenous RNAs with extended regions of sequence identity to the trigger (see Fire, 1999 for a review). A striking feature of dsRNA-triggered genetic interference processes (RNAi) in C. elegans is the ability of the interference to ‘spread’ to cells that are some distance from the initial site of dsRNA delivery. The first indication of this spreading effect came from the observation that dsRNA injected into the body cavity produced interference phenotypes throughout the injected animal (Fire et al., 1998). A later and more dramatic demonstration of a spreading effect was observed after feeding animals food that contained dsRNA (Timmons and Fire, 1998) or after soaking animals in dsRNA (Tabara et al., 1998).

The manner in which an organism responds to nucleic acid encountered in food is of interest from biological and technical perspectives. The predominant component of the C. elegans diet in the laboratory is bacteria; therefore, introduction of dsRNA into the C. elegans diet has involved engineering bacteria to produce dsRNA (Timmons and Fire, 1998). Although specific interference for several genes was evident in earlier studies with dsRNA-producing bacteria (Timmons and Fire, 1998), the observed phenotypes were limited in penetrance and expressivity. While demonstrating that ingested dsRNA is competent to trigger interference, these initial experiments left unresolved the question of whether loss-of-function phenotypes could be effectively produced by ingested dsRNA. To address this question, we sought to increase the effectiveness of the bacterial feeding technique by several means, including modifications that increase the concentration of dsRNA in the bacterial cell. We have found that a bacterial strain lacking the dsRNA-specific endonuclease RNaseIII can be cultured to produce high levels of specific dsRNA and that these bacteria can effectively trigger strong and gene-specific phenotypic responses when fed to C. elegans.

Section snippets

C. elegans strains

All C. elegans strains were derived from the wild-type N2 strain of Brenner (Brenner, 1974). Three transgenic gfp strains were used in experiments for which interference with gfp was tested: PD4251 (ccIs4251) is homozygous for a myo-3::gfp transgene insertion (Fire et al., 1998); VH41 is homozygous for an unc-119::gfp transgene insertion (rhIs13) (a gift from Dr H. Hutter, Max Planck); and PD8047 harbors an extrachromosomal array with a let-858::gfp reporter (ccEx8047) (Kelly et al., 1997). him

Assays for bacteria-induced RNA interference

In each experiment, an engineered bacterial strain was provided as the sole source of food for C. elegans. The strains had been transformed with a single plasmid designed to express a specific segment of dsRNA. The bacterial food was provided to worms on standard nematode culture plates (+ additives) (Brenner, 1974). Interference phenotypes were monitored in the F1 progeny of animals placed on the modified food. In general, the levels of interference obtained with modified bacterial strains

Conclusions

We have shown that continuous feeding of C. elegans with bacteria that have been engineered to produce high levels of dsRNA can yield animals with loss-of-function phenotypes for a variety of target genes. Our data suggests, given several caveats, that an accurate indication of functional roles of genes for which loss-of-function mutations are not available can be achieved by comparisons of RNAi phenotypes from dsRNA injection and feeding techniques. We have observed specific limitations in RNA

Acknowledgements

We thank N. Costantino, H. Ellis, J. Fleenor, M. Hsu, H. Hutter, C. McGill, P. Newmark, M. Nonet, and J. Yanowitz for their help and suggestions. This work was supported by PHS grants R01GM37706 (AF) and F32HD08353 (LT).

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