Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans - PubMed (original) (raw)
Comparative Study
Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans
R S Kamath et al. Genome Biol. 2001.
Abstract
Background: In Caenorhabditis elegans, injection of double-stranded RNA (dsRNA) results in the specific inactivation of genes containing homologous sequences, a technique termed RNA-mediated interference (RNAi). It has previously been shown that RNAi can also be achieved by feeding worms Escherichia coli expressing dsRNA corresponding to a specific gene; this mode of dsRNA introduction is conventionally considered to be less efficient than direct injection, however, and has therefore seen limited use, even though it is considerably less labor-intensive.
Results: Here we present an optimized feeding method that results in phenotypes at least as strong as those produced by direct injection of dsRNA for embryonic lethal genes, and stronger for genes with post-embryonic phenotypes. In addition, the interference effect generated by feeding can be titrated to uncover a series of hypomorphic phenotypes informative about the functions of a given gene. Using this method, we screened 86 random genes on consecutive cosmids and identified functions for 13 new genes. These included two genes producing an uncoordinated phenotype (a previously uncharacterized POU homeodomain gene, ceh-6, and a gene encoding a MADS-box protein) and one gene encoding a novel protein that results in a high-incidence-of-males phenotype.
Conclusions: RNAi by feeding can provide significant information about the functions of an individual gene beyond that provided by injection. Moreover, it can be used for special applications for which injection or the use of mutants is sometimes impracticable (for example, titration, biochemistry and large-scale screening). Thus, RNAi by feeding should make possible new experimental approaches for the use of genomic sequence information.
Figures
Figure 1
L4440 double-T7 vector inside HT115 RNase-deficient E. coli. A fragment from the gene of interest is amplified by PCR and cloned into the L4440 double-T7 vector, which has two T7 promoters in inverted orientation flanking the multiple cloning site [4]. Cloned plasmids are transformed into HT115(DE3), an RNase III-deficient E. coli strain with IPTG-inducible expression of T7 polymerase (L. Timmons and A. Fire, personal communication).
Figure 2
Tissue susceptibility to RNAi by feeding. Worms with a transgenic GFP reporter gene expressed in all somatic tissues (egl-27::gfp) were fed (a) non-dsRNA expressing bacteria or (b-e) bacteria expressing gfp dsRNA for (b) 24 h, (c) 48 h, (d) 72 h, or (e) 96 h. After being fed for 24 h, GFP expression was markedly reduced (b) compared to similarly treated unfed worms (a). (c) Of worms fed for 48 h, none had any visible non-neural GFP expression, and 20/22 (91%) had reduced levels of neural GFP. (d) After 72 h, 93% had no non-neural somatic GFP expression (the remaining 7% had very weak expression), and 28/30 (93%) had reduced neural GFP. (e) Finally, after being fed for 96 h, no worms had any non-neural GFP expression, and 26/27 (96%) also had reduced levels of neural GFP compared with similarly treated unfed worms. The head of each worm (from the nose to the posterior pharynx), which contains the majority of the neurons in the animal, is indicated with a bracket.
Figure 3
Embryonic phenotypes from RNAi by feeding versus injection. (a) Wild-type N2 embryos divide with the AB (anterior cell) spindle oriented along the dorso-ventral axis and the P1 (posterior cell) spindle along the AP axis to create a four-cell embryo (d). In wild-type embryos, AB is larger and divides slightly before P1. For par-2, RNAi by both (b) feeding and (c) injection results in embryos in which AB and P1 are of equal size and divide synchronously, with both spindles oriented along the dorso-ventral axis. Embryos in (a-c) are undergoing the second mitotic divisions. For bir-1, RNAi by both (e) feeding and (f) injection results in embryos that do not complete cytokinesis and thus form a single multinucleate cell. Embryos in (d-f) have undergone two rounds of mitosis. Anterior is to the left in all panels.
Figure 4
Large-scale comparison of RNAi by feeding versus injection. The first 1,200 predicted genes from chromosome I were screened by feeding [21]. Those genes with a phenotype were subjected to RNAi by injection, and results obtained by the two methods were compared according to phenotypic class - embryonic lethal (Emb), sterile (Ste), or post-embryonic (PostEmb; see the Materials and methods section for scoring criteria). Data shown compare results from three worms subjected to RNAi by feeding or injection.
References
- Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998;391:806–811. - PubMed
- Bass BL. Double-stranded RNA as a template for gene silencing. Cell. 2000;101:235–238. - PubMed
- Timmons L, Fire A. Specific interference by ingested dsRNA. Nature. 1998;395:854. - PubMed
- Hunter CP. Genetics: a touch of elegance with RNAi. Curr Biol. 1999;9:R440–R442. - PubMed
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