“Bacterially produced double-stranded RNA is consumed by Caenorhabditis elegans and enters the endogenous RNA interference pathway to reduce expression of the corresponding target gene.”

This was further demonstrated by the fact that ‘environmental RNAi’, soaking animals in dsRNA or feeding animals bacteria expressing dsRNA, allowed RNAi transmission through the intestine, inducing potent silencing in most tissues [2,3]. … During environmental RNAi, ingested dsRNA (double black line) enters the worm via SID-2 (blue) on the intestinal lumen. SID-5 (red) is associated with endosomes (white circles) and is required for dsRNA transit and/or exit from the intestine into the pseudocoelom (body cavity). SID-1 (purple) and SID-3 (yellow) are required to import dsRNA back into the intestine or into more distal tissues, such as the body wall muscle cells, to suppress expression of a target gene (green). … Similar to sid-1 and sid-3, sid-5 is required for environmental RNAi targeting both exogenously expressed green fluorescent protein and endogenous targets [12].… One of the phenomena discovered early on was that microinjection of dsRNA into one part of the body elicited silencing in another part, evoking the existence of an RNA-transport system. This was further demonstrated by the fact that ‘environmental RNAi’, soaking animals in dsRNA or feeding animals bacteria expressing dsRNA, allowed RNAi transmission through the intestine, inducing potent silencing in most tissues [2,3]. The best understood mediator of systemic RNAi in C. elegans is SID-1, a dsRNA membrane channel required for cellular import of RNAi silencing signals [4–6]. … Expression of sid-1(+) in the body wall muscle cells of a sid-1(-) animal restores RNAi specifically in the body wall muscles in response to both environmental RNAi transiting the intestine and transgenic RNAi expressed in the pharynx (Figure 1) [4,8]. Figure 1 Steps of dsRNA transport in C. elegans. Show full caption During environmental RNAi, ingested dsRNA (double black line) enters the worm via SID-2 (blue) on the intestinal lumen. SID-5 (red) is associated with endosomes (white circles) and is required for dsRNA transit and/or exit from the intestine into the pseudocoelom (body cavity). SID-1 (purple) and SID-3 (yellow) are required to import dsRNA back into the intestine or into more distal tissues, such as the body wall muscle cells, to suppress expression of a target gene (green). Similarly, during transgenic RNAi, SID-5 is likely required for export of the silencing signal (depicted as a double-stranded RNA hairpin) out of the cell, in this case the pharynx, while SID-1 and SID-3 are required for import in distal tissues. … sid-5 mutants were identified in the same genetic screens that identified mutations in the sid-1, sid-2 and sid-3 genes [4]; however, sid-5 has a distinct role in systemic RNAi. The authors' work suggests that SID-5 regulates cellular export of RNAi silencing signals. Similar to sid-1 and sid-3, sid-5 is required for environmental RNAi targeting both exogenously expressed green fluorescent protein and endogenous targets [12]. The sid-5 gene is required for the spreading of transgene-derived RNAi silencing signals from the pharynx to the body wall muscle cells, but is not required for the transgene to elicit cell autonomous RNAi in the pharynx. …

Furthermore, in C. elegans, dsRNA present in the environment or expressed in ingested bacteria is sufficient to trigger RNAi (environmental RNAi). … sid-2(qt13) worms were completely resistant to bacteria-mediated RNAi targeting endogenous somatic and germ-line-expressed genes (Tables 1 and 2 and data not shown) and strongly resistant to soaking-mediated RNAi of the same genes. However, similar to wild-type and in contrast to sid-1 mutants, sid-2(qt13) worms were fully sensitive to systemic RNAi initiated by injection or transgenic expression of dsRNA targeting somatic and germ-line-expressed genes (Tables 1 and 2; Fig. 1). Thus, sid-2 is required for the uptake of environmental dsRNA but, unlike sid-1, is not required for the subsequent spread of dsRNA between cells. … Our results delineate two steps in systemic RNAi, whereby sid-2 functions in the intestine to bring dsRNA into the animal, and sid-1 functions to distribute dsRNA into peripheral cells.Abstract In plants and in the nematode Caenorhabditis elegans, an RNAi signal can trigger gene silencing in cells distant from the site where silencing is initiated. In plants, this signal is known to be a form of dsRNA, and the signal is most likely a form of dsRNA in C. elegans as well. Furthermore, in C. elegans, dsRNA present in the environment or expressed in ingested bacteria is sufficient to trigger RNAi (environmental RNAi). Ingestion and soaking delivery of dsRNA has also been described for other invertebrates. Here we report the identification and characterization of SID-2, an intestinal luminal transmembrane protein required for environmental RNAi in C. elegans. … sid-2 mutants were isolated as animals resistant to bacteria-mediated systemic RNAi of a GFP reporter but sensitive to transgene-mediated systemic RNAi of the same reporter (7). One sid-2 mutant (qt13) was selected for initial analysis, mapping, and cloning. We found that sid-2(qt13) worms were completely resistant to bacteria-mediated RNAi targeting endogenous somatic and germ-line-expressed genes (Tables 1 and 2 and data not shown) and strongly resistant to soaking-mediated RNAi of the same genes. However, similar to wild-type and in contrast to sid-1 mutants, sid-2(qt13) worms were fully sensitive to systemic RNAi initiated by injection or transgenic expression of dsRNA targeting somatic and germ-line-expressed genes (Tables 1 and 2; Fig. 1). Thus, sid-2 is required for the uptake of environmental dsRNA but, unlike sid-1, is not required for the subsequent spread of dsRNA between cells. Characterization of sid-2 deficiency on RNAi of a germ-line-expressed gene … ## Conclusions Our results delineate two steps in systemic RNAi, whereby sid-2 functions in the intestine to bring dsRNA into the animal, and sid-1 functions to distribute dsRNA into peripheral cells. However, sid-1 is also required for environmental RNAi-mediated silencing in intestinal cells. Therefore, it is probable that SID-2 does not directly mediate transport of dsRNA into the cytoplasm of intestinal cells. …

Here we show that C. elegans can respond in a gene-specific manner to dsRNA encountered in the environment. C. elegans normally feed on bacteria, ingesting and grinding them in the pharynx and subsequently absorbing bacterial contents in the gut. We find that Escherichia coli bacteria expressing dsRNAs can confer specific interference effects on the nematode larvae that feed on them.Abstract A genetic interference phenomenon in the nematode Caenorhabditis elegans has been described in which expression of an individual gene can be specifically reduced by microinjecting a corresponding fragment of double-stranded (ds) RNA 1. One striking feature of this process is a spreading effect: interference in a broad region of the animal is observed following the injection of dsRNA into the extracellular body cavity. Here we show that C. elegans can respond in a gene-specific manner to dsRNA encountered in the environment. C. elegans normally feed on bacteria, ingesting and grinding them in the pharynx and subsequently absorbing bacterial contents in the gut. We find that Escherichia coli bacteria expressing dsRNAs can confer specific interference effects on the nematode larvae that feed on them. This is a preview of subscription content, access via your institution

Andrew Fire and Craig Mello labs later developed alternative ways for triggering RNAi in C. elegans with exogenous dsRNAs [2, 3] . What they did was soaking worms in dsRNA solution or feeding them with recombinant E. coli engineered to express dsRNA (feeding RNAi) [2, 3] . These findings indicated that exogenous RNA, specifically dsRNA, could enter cells of C. elegans and initiate downstream RNAi effects. … Once inside C. elegans cells, exogenous dsRNAs follow a similar pathway to endogenous RNAi. Basically these dsR-NAs are cut by DCR-1 with the assistance of RDE-4, RDE-1, DRH-1, and DRH-2 [18] [19] [20] [21] [22] (Figure 2 ). RDE-1 then directs the so-called primary siRNA to its targeting mRNA, resulting in the degradation of target mRNA and triggering the amplification of secondary siRNA by RNA dependent RNA polymerases (RdRP) such as RRF-1 and EGO-1 with the involvement of DRH-3 [23] [24] [25] [26] [27] .… Here we highlight some known examples of environmental RNA interference (RNAi) in animals and introduce the molecular mechanisms underneath. Publication of the landmark article by Andrew Fire, Craig Mello, and colleagues in 1998 has linked double-stranded RNA (dsRNA) with the RNA interference (RNAi) phenomenon [1] . DsRNAs targeting specific gene sequences were injected into Caenorhabditis elegans for this work [1] . Andrew Fire and Craig Mello labs later developed alternative ways for triggering RNAi in C. elegans with exogenous dsRNAs [2, 3] . What they did was soaking worms in dsRNA solution or feeding them with recombinant E. coli engineered to express dsRNA (feeding RNAi) [2, 3] . These findings indicated that exogenous RNA, specifically dsRNA, could enter cells of C. elegans and initiate downstream RNAi effects. The fact that dsRNAs both injected into and fed to worms could trigger a systemic RNAi effect in most cells also indicated the existence of a spreading mechanism for cell to cell transportation/uptake of RNAi signals, presumably also dsRNAs. Actually this kind of environmental and cell nonautonomous RNAi effects have now been found in many organisms including animals, plants, and protozoans [4] [5] [6] . … ## elegans RNAi pathway triggered by exogenous RNA Once inside C. elegans cells, exogenous dsRNAs follow a similar pathway to endogenous RNAi. Basically these dsR-NAs are cut by DCR-1 with the assistance of RDE-4, RDE-1, DRH-1, and DRH-2 [18] [19] [20] [21] [22] (Figure 2 ). RDE-1 then directs the so-called primary siRNA to its targeting mRNA, resulting in the degradation of target mRNA and triggering the amplification of secondary siRNA by RNA dependent RNA polymerases (RdRP) such as RRF-1 and EGO-1 with the involvement of DRH-3 [23] [24] [25] [26] [27] . CSR-1 and some other secondary Argonaute proteins (SAGO) may be responsible for the gene silencing effect of secondary siRNA, and a number of factors such as MUT-7/-14/-15/-16, RDE-2/-3, SAGO-1/-2, and PPW-1 may play crucial roles in the effect of secondary siRNA [28] [29] [30] [31] [32] (Figure 2 ). Nine out of 61 C. elegans ATP-binding cassette (ABC) transporters are also involved in the environmental RNAi effect [33] . …

Double-stranded RNA interference (dsRNAi) provides an alternative means to identify genes involved in important biological processes. In this method, dsRNA matching the coding sequence of an endogenous gene is introduced into the worm. The dsRNA is processed in vivo to generate small (21-22 nucleotide) RNAs that serve as guides to find and bind the matching endogenous mRNAs, targeting them for destruction 1 . … Introduction of dsRNAs into an animal can be achieved by direct injection of dsRNA 2 , by soaking animals in dsRNA 3 , or by feeding animals bacteria that express dsRNA 4 . Each of these delivery methods cause systemic knockdown effects as most cells of the animal express SID-1, a transmembrane channel capable of importing dsRNA 5 . … When we fed L1 stage eri-1; lin15B larvae bacteria expressing egl-30 dsRNA, we found that the larvae grew to become adults that showed clear defects in locomotion and egg-laying behavior. After three days, 100% of the animals fed dsRNA against egl-30 were paralyzed and unable to lay eggs (Figures 2B, Table 1 ).… There are many advantages to performing such chemical mutagenesis screens as they create permanent lesions within the worm genome that can result in both gain-of-function and loss-of-function phenotypes, but the mapping procedure is slow and expensive. Double-stranded RNA interference (dsRNAi) provides an alternative means to identify genes involved in important biological processes. In this method, dsRNA matching the coding sequence of an endogenous gene is introduced into the worm. The dsRNA is processed in vivo to generate small (21-22 nucleotide) RNAs that serve as guides to find and bind the matching endogenous mRNAs, targeting them for destruction 1 . This procedure is relatively quick, but because dsRNA targets mRNA rather than DNA, only reduction-of-function phenotypes are observed using this approach. Introduction of dsRNAs into an animal can be achieved by direct injection of dsRNA 2 , by soaking animals in dsRNA 3 , or by feeding animals bacteria that express dsRNA 4 . Each of these delivery methods cause systemic knockdown effects as most cells of the animal express SID-1, a transmembrane channel capable of importing dsRNA 5 . Originally used as a reverse genetics approach to knockdown the expression of individual genes one at a time, the development of two feeding libraries now permits large-scale genetic screens. The commercially-available dsRNA libraries contain bacterial clones representing either 55% or 87% of all C. elegans genes 6, 7 . … egl-30, encodes the C. elegans ortholog of the G protein subunit Gaq. egl-30 null mutants arrest during early larval development, but some null escapers and hypomorphic loss-of-function egl-30 mutants grow to become adult stage and are defective in locomotion and egg-laying behaviors 20 . When we fed L1 stage eri-1; lin15B larvae bacteria expressing egl-30 dsRNA, we found that the larvae grew to become adults that showed clear defects in locomotion and egg-laying behavior. After three days, 100% of the animals fed dsRNA against egl-30 were paralyzed and unable to lay eggs (Figures 2B, Table 1 ). We did not observe the larval arrest phenotype in our dsRNA knockdown experiments that is observed in egl-30(ad810) null animals. This is presumably because the L1 animals we plated onto egl-30 dsRNA bacteria had already passed the early developmental requirement for EGL-30. …

RNAi can be achieved by feeding worms Escherichia coli expressing dousble-stranded RNA corresponding to a specific gene. … 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 … RNA-mediated interference (RNAi) is the phenomenon first described in the nematode Caenorhabditis elegans in which introduction of double-stranded RNA (dsRNA) results in potent and specific inactivation of the corresponding gene through the degradation of endogenous mRNAShort abstract RNAi can be achieved by feeding worms Escherichia coli expressing dousble-stranded RNA corresponding to a specific gene. An optimized feeding method is presented that results in phenotypes at least as strong as those produced by direct injection of RNA for embryonic lethal genes, and stronger for genes with post-embryonic phenotypes. 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 … ## Background RNA-mediated interference (RNAi) is the phenomenon first described in the nematode Caenorhabditis elegans in which introduction of double-stranded RNA (dsRNA) results in potent and specific inactivation of the corresponding gene through the degradation of endogenous mRNA [1,2]. This technique rapidly produces gene-specific loss-of-function or hypomorphic phenotypes, and potent interference is also observed in the progeny of the affected animal. …

In 1998, Fire and Mello discovered that injection of double stranded RNA (dsRNA) into worms leads to specific degradation of the corresponding mRNA, a process termed RNA interference (RNAi; Fire et al., 1998). Soon afterwards, it was found that either soaking worms in dsRNA solution or feeding worms bacteria engineered to produce dsRNA also could induce a robust RNAi response (Tabara et al., 1998; Timmons and Fire, 1998). … RNAi by feeding: bacteria producing the desired dsRNA are fed to worms and either they or their progeny are scored.Julie Ahringer, The Gurdon Institute, University of Cambridge, Cambridge CB2 1QN, UK In 1998, Fire and Mello discovered that injection of double stranded RNA (dsRNA) into worms leads to specific degradation of the corresponding mRNA, a process termed RNA interference (RNAi; Fire et al., 1998). Soon afterwards, it was found that either soaking worms in dsRNA solution or feeding worms bacteria engineered to produce dsRNA also could induce a robust RNAi response (Tabara et al., 1998; Timmons and Fire, 1998). The technique of RNAi, coupled with the availability of the complete genomic sequence of C. elegans (Consortium, 1998), has made possible the rapid study of gene function, both on a single gene level and at a global scale. This section discusses general features to consider when using this technique. Sections following this give detailed protocols for RNAi by injection, soaking and feeding. 2.1. Which RNAi method to use? … RNAi by soaking: worms are soaked in a high concentration dsRNA solution and then subsequently they or their progeny scored for phenotypes. RNAi by soaking is useful for treating a moderately large number of animals (e.g., tens to hundreds) or for high throughput screening in 96 well format. Worms of any stage can be soaked. More dsRNA is needed for soaking than for injection (5–10 μg per experiment). RNAi by feeding: bacteria producing the desired dsRNA are fed to worms and either they or their progeny are scored. RNAi by feeding is the least labor intensive and most inexpensive method, but produces slightly more variable results than RNAi by soaking or injection. RNAi by feeding can be used to treat a large number of animals at once or for high throughput screening, both on agar plates and in liquid culture. Worms of any stage can be subjected to RNAi by feeding. 2.2. Which worm strain to use?

These results suggest that dsRNA against GFP is produced by B. subtilis and is capable of an effective gene-specific knock-down of GFP expression in C. elegans upon ingestion. … First, the daf-2 (RNAi) and glp-1 (RNAi) treatments in wild type worms lead to approximately 20 to 40% decrease in the corresponding mRNA levels (S2B Fig). … In summary, we designed a second system that would allow continuous feeding of C. elegans with B. subtilis bacteria that have been engineered to produce dsRNA against various worm target genes. As proof of principal, we tested this RNAi system to produce animals with loss-of-function phenotypes for a small number of target genes previously shown to be involved in aging.… GFP expression levels were reduced to 3% of the original expression level in the DAF-16::GFP worms, and were lowered almost 6 times in SUR-5::GFP animals. As on E. coli, B. subtilis expressing dsRNA was not able to affect neuronal GFP expression (Fig 2). These results suggest that dsRNA against GFP is produced by B. subtilis and is capable of an effective gene-specific knock-down of GFP expression in C. elegans upon ingestion. Effect of B. subtilis induced RNAi against known regulators of longevity on DAF-16 nuclear localization … Clones that tested positive by PCR analysis and restriction digestion were transformed into the B. subtilis BG322 strain for further analysis. The efficiency of RNAi-mediated down-regulation of these genes was tested by qRT-PCR and by scoring previously described phenotypes. First, the daf-2 (RNAi) and glp-1 (RNAi) treatments in wild type worms lead to approximately 20 to 40% decrease in the corresponding mRNA levels (S2B Fig). The daf-2 (RNAi) and glp-1 (RNAi) treatments in RNAi-sensitive (NL2099) worms lead to approximately 40 to 50% decrease in the corresponding mRNA levels (data not shown). Unfortunately we were unable to detect a significant reduction in unc-62 level of expression. … subtilis promoted health span extension? More experiments are needed to address these issues. It is entirely possible that we can use B. subtilis and C. elegans to identify a novel mechanism of longevity in worms by examining the beneficial effects of food on organ (intestine in this case) physiology. In summary, we designed a second system that would allow continuous feeding of C. elegans with B. subtilis bacteria that have been engineered to produce dsRNA against various worm target genes. As proof of principal, we tested this RNAi system to produce animals with loss-of-function phenotypes for a small number of target genes previously shown to be involved in aging. These experiments showed first the effectiveness of this alternative method, and second, highlighted phenotypic differences that depend on which bacterial host is being used to deliver the dsRNAs molecules. …

These results suggest that dsRNA against GFP is produced by B. subtilis and is capable of an effective gene-specific knock-down of GFP expression in C. elegans upon ingestion. … First, the daf-2 (RNAi) and glp-1 (RNAi) treatments in wild type worms lead to approximately 20 to 40% decrease in the corresponding mRNA levels (S2B Fig). … In summary, we designed a second system that would allow continuous feeding of C. elegans with B. subtilis bacteria that have been engineered to produce dsRNA against various worm target genes. As proof of principal, we tested this RNAi system to produce animals with loss-of-function phenotypes for a small number of target genes previously shown to be involved in aging.… GFP expression levels were reduced to 3% of the original expression level in the DAF-16::GFP worms, and were lowered almost 6 times in SUR-5::GFP animals. As on E. coli, B. subtilis expressing dsRNA was not able to affect neuronal GFP expression (Fig 2). These results suggest that dsRNA against GFP is produced by B. subtilis and is capable of an effective gene-specific knock-down of GFP expression in C. elegans upon ingestion. Effect of B. subtilis induced RNAi against known regulators of longevity on DAF-16 nuclear localization … Clones that tested positive by PCR analysis and restriction digestion were transformed into the B. subtilis BG322 strain for further analysis. The efficiency of RNAi-mediated down-regulation of these genes was tested by qRT-PCR and by scoring previously described phenotypes. First, the daf-2 (RNAi) and glp-1 (RNAi) treatments in wild type worms lead to approximately 20 to 40% decrease in the corresponding mRNA levels (S2B Fig). The daf-2 (RNAi) and glp-1 (RNAi) treatments in RNAi-sensitive (NL2099) worms lead to approximately 40 to 50% decrease in the corresponding mRNA levels (data not shown). Unfortunately we were unable to detect a significant reduction in unc-62 level of expression. … subtilis promoted health span extension? More experiments are needed to address these issues. It is entirely possible that we can use B. subtilis and C. elegans to identify a novel mechanism of longevity in worms by examining the beneficial effects of food on organ (intestine in this case) physiology. In summary, we designed a second system that would allow continuous feeding of C. elegans with B. subtilis bacteria that have been engineered to produce dsRNA against various worm target genes. As proof of principal, we tested this RNAi system to produce animals with loss-of-function phenotypes for a small number of target genes previously shown to be involved in aging. These experiments showed first the effectiveness of this alternative method, and second, highlighted phenotypic differences that depend on which bacterial host is being used to deliver the dsRNAs molecules. …

We expressed SID-1, a transmembrane protein from Caenorhabditis elegans that is required for systemic RNA interference (RNAi), in C. elegans neurons. This expression increased the response of neurons to double-stranded (ds)RNA delivered by feeding. … Worms expressing neuronal SID-1 showed RNAi phenotypes when fed with bacteria expressing dsRNA for known neuronal genes and for uncharacterized genes with no previously known neuronal phenotypes.Abstract We expressed SID-1, a transmembrane protein from Caenorhabditis elegans that is required for systemic RNA interference (RNAi), in C. elegans neurons. This expression increased the response of neurons to double-stranded (ds)RNA delivered by feeding. Mutations in the lin-15b and lin-35 genes enhanced this effect. Worms expressing neuronal SID-1 showed RNAi phenotypes when fed with bacteria expressing dsRNA for known neuronal genes and for uncharacterized genes with no previously known neuronal phenotypes. Neuronal expression of sid-1 decreased nonneuronal RNAi, suggesting that neurons expressing transgenic sid-1(+) served as a sink for dsRNA. This effect, or a sid-1(–) background, can be used to uncover neuronal defects for lethal genes. Expression of sid-1(+) from cell-specific promoters in sid-1 mutants results in cell-specific feeding RNAi. We used these strains to identify a role for integrin signaling genes in mechanosensation.

In the feeding protocol, dsRNA is transcribed in E. coli and ingested by animals. … RNAi can be induced by feeding worms bacteria expressing dsRNA (Timmons et al., 2001). … RNA interference (RNAi) is a form of sequence-specific, post-transcriptional gene silencing (PTGS) that is triggered by the introduction of dsRNA (unit 26.1; Fire et al., 1998).… In the microinjection and soaking protocols, in vitro preparations of dsRNA are delivered mechanically using a needle to inject dsRNA directly into the body or passively by soaking worms in dsRNA solution. In the feeding protocol, dsRNA is transcribed in E. coli and ingested by animals. The protocols provided in this unit are meant to help the complete novice at worm culture select and apply the appropriate RNAi delivery method. Towards that end, protocols are also provided for transferring and propagating (see Support Protocol 1) and for sexing and mating (see Support Protocol 2) C. elegans. … ### Feeding Worms dsRNA RNAi can be induced by feeding worms bacteria expressing dsRNA (Timmons et al., 2001). First, a cDNA corresponding to the gene of interest is cloned into a bacterial expression vector between opposing phage T7 polymerase promoter sites. The feeding vector is then transformed into the E. coli HT115 strain carrying the DE3 lysogen (unit 1.8), providing IPTG inducible expression of the phage T7 RNA polymerase. … #### Background Information RNA interference (RNAi) is a form of sequence-specific, post-transcriptional gene silencing (PTGS) that is triggered by the introduction of dsRNA (unit 26.1; Fire et al., 1998). RNAi is related to a number of gene-silencing phenomena in a variety of organisms. Genetic and biochemical studies have revealed that the underlying mechanisms of these silencing phenomena are remarkably conserved (for reviews, see unit 26.1 and Hannon, 2002). …

The worms eat the bacteria, … The induced bacteria are fed to the worms, which absorb the dsRNA, and the F1 generation are analysed for their RNAi phenotype, often under the dissecting scope. The fictional example shows that upon silencing of the gene xyz-1 by RNAi, a change in crawling behaviour is observed. … RNAi most commonly leads to a knockdown rather than a knockout effect.Since C. elegans feeds on lawns of E. coli, it was simple to replace the usual strain with a transformed strain engineered to transcribe a cloned gene from both the sense and antisense directions, producing dsRNA of a specific gene (Figure 1 ). The worms eat the bacteria, teria, thereby impairing dsRNA production, in the vast majority of cases RNAi can be performed by feeding. From these three different options, the experimentalist can choose the most appropriate according to their needs. … (B) Induction by IPTG to produce dsRNA. To produce dsRNA, the bacteria have to be exposed to IPTG, this will induce the production of T7 polymerase, which will in turn produce the dsRNA. (C) Ingestion of bacteria, spreading of RNAi effect and scoring of the F1 progeny. The induced bacteria are fed to the worms, which absorb the dsRNA, and the F1 generation are analysed for their RNAi phenotype, often under the dissecting scope. The fictional example shows that upon silencing of the gene xyz-1 by RNAi, a change in crawling behaviour is observed. and its role has been studied extensively in vulva development. Approximately ten different alleles exist for let-60 17 , the Ras orthologue in C. elegans 18-20 . Some are gain-of-function alleles, producing a multiple vulvae (Muv) phenotype, and others are loss-offunction, producing a vulvaless (Vul) phenotype (Figure 3). … D.P.W. is supported by a Wellcome Trust Studentship. We are also grateful to Verena Wolfram for comments on the manuscript. observed in the same progeny population. Therefore it should be kept in mind that RNAi most commonly leads to a knockdown rather than a knockout effect. Another problem encountered with RNAi is specificity. Since we know that RNAi-mediated silencing specificity depends upon the RNA sequence, it is prudent to set a limit of not more than 80% identity over 200 bp with another gene, to ensure specificity 7 . The next limitation is the restriction to loss-of-function phenotypes. …

After 48 hours, with the exception of the nervous system, GFP was not detectable in somatic tissues; furthermore, neural GFP was dramatically reduced in 91% of worms (n = 22; Figure 2c ). A similar level of inhibition of GFP expression was observed after 72 and 96 hours of feeding (Figure 2d, e ). … In summary, RNAi by feeding efficiently silences genes in most C. elegans somatic tissues; however, the nervous system has a delayed and somewhat less robust response to RNAi compared to other tissues. … We present a method by which RNAi by feeding is as strong and as sensitive as RNAi by injection for detecting embryonic phenotypes, and, furthermore, is more sensitive for detecting sterility and post-embryonic phenotypes.… After being fed for 24 hours at 15°C, GFP expression was markedly reduced compared to similarly treated unfed worms (n = 28; compare Figure 2b and a ). After 48 hours, with the exception of the nervous system, GFP was not detectable in somatic tissues; furthermore, neural GFP was dramatically reduced in 91% of worms (n = 22; Figure 2c ). A similar level of inhibition of GFP expression was observed after 72 and 96 hours of feeding (Figure 2d, e ). Although GFP fluorescence in fed worms was abolished or severely reduced, GFP was sometimes expressed at high levels in late-stage embryos derived from these worms (data not shown). This suggests that some zygotic embryonic transcripts may be difficult to silence, possibly because a continuous supply of dsRNA cannot be provided through the eggshell. In summary, RNAi by feeding efficiently silences genes in most C. elegans somatic tissues; however, the nervous system has a delayed and somewhat less robust response to RNAi compared to other tissues. Comparison of RNAi by feeding and injection for maternal-effect lethal genes … ### Discussion We present a method by which RNAi by feeding is as strong and as sensitive as RNAi by injection for detecting embryonic phenotypes, and, furthermore, is more sensitive for detecting sterility and post-embryonic phenotypes. The fact that feeding is superior to injection for detecting sterile phenotypes is most likely due to the fact that with feeding the RNA interference effect is begun at L4 stage, giving it more time to affect the germline or gonad, whereas young adults are usually injected; nevertheless, this does not diminish the utility of …

In this RNAi, bacteria producing desired dsRNA (RNAi bacteria) are fed to animals, and either they or their progeny are scored for mutant phenotypes. The effect of the RNAi continues during feeding of the bacteria. … These results indicate that oma-1;oma-2 RNAi significantly decreased the corresponding transcripts simultaneously. … When the tested animals were subjected to the control RNAi using the empty vector, GFP fluorescence and mature oocytes in the uterus were observed, as shown in Fig. 3A. Under daf-16 RNAi and under gfp RNAi, almost all the fluorescence of GFP disappeared (Fig. 3B, C), indicating that both RNAi suppressed the expression of GFP, as expected.… RNAi, knockdown of gene’s function, was first performed in the nematode Caenorhabditis elegans. 1) In C. elegans, there are three ways to introduce double-stranded RNA (dsRNA) for induction of RNAi: microinjection, soak ing, and feeding. 1–3) Among these, feeding RNAi is the least labor-intensive and most inexpensive method. In this RNAi, bacteria producing desired dsRNA (RNAi bacteria) are fed to animals, and either they or their progeny are scored for mutant phenotypes. The effect of the RNAi continues during feeding of the bacteria. Hence, it can be used to measure lifespan, which is necessary for long-term observation. 4,5) When knock down of two genes (double knockdown) is carried out, injection RNAi or soaking RNAi is usually adopted. … 1C). However, under the double feeding, the amounts of oma-1 and oma-2 mRNAs decreased to 2-fold of control RNAi. In contrast, under oma-1;oma-2 RNAi (double knock down), the amounts of oma-1 and oma-2 mRNAs decreased to 7- and 16-fold of control RNAi. These results indicate that oma-1;oma-2 RNAi significantly decreased the corresponding transcripts simultaneously. -primed cDNAs, and Thunderbird SYBR qPCR Mix (Toyobo, Osaka, Japan). qPCR was run and monitored on a LineGene real-time thermal cycler (BioFlux, Tokyo) under the following reaction conditions: 95 C for 15 s, 55 C for 15 s, and 72 C for 30 s (40 cycles). … The plasmid pPD-Venus encoding an altered type of GFP was used as a template in this amplification (162–997 bp). Then we constructed the RNAi plasmid for gfp and that for gfp in addition to oma-1 and oma-2, as described above (Fig. 1A). When the tested animals were subjected to the control RNAi using the empty vector, GFP fluorescence and mature oocytes in the uterus were observed, as shown in Fig. 3A. Under daf-16 RNAi and under gfp RNAi, almost all the fluorescence of GFP disappeared (Fig. 3B, C), indicating that both RNAi suppressed the expression of GFP, as expected. Because the daf-16 RNAi induced degradation of the mRNA, including both the daf-16 and the gfp region, degradation resulted in the death of a portion of gfp. The RNAi showed no influence on production of the mature oocytes. …

Entry of extracellular dsRNA into the cytosol and subsequent silencing relies on the conserved dsRNA importer SID-1 … Upon recognition of a matching mRNA by RDE-1-bound siRNAs, RNA-dependent RNA Polymerases (RdRPs) are recruited, resulting in the production of numerous secondary siRNAs … Through these events, extracellular dsRNA can reduce the levels of mRNA and/or pre-mRNA of a target gene.… Studies in C. elegans have therefore been informative in piecing together the organismal response to RNAi in an animal. While similar silencing responses occur in diverse cell types, it is unclear whether dsRNA from every source engages the same molecular machinery in each cell. Entry of extracellular dsRNA into the cytosol and subsequent silencing relies on the conserved dsRNA importer SID-1 (7) (8) (9) (10) . SID-1-dependent silencing is observed in many tissues even when dsRNA is expressed within a single tissue, suggesting that form(s) of dsRNA move between cells. … Silencing in these diverse target cells requires the dsRNA-binding protein RDE-4 (15, 16) and the endonuclease DCR-1, which together process dsRNA into small-interfering RNAs (siRNAs) (17, 18) , and the Argonaute RDE-1, which binds siRNAs (19) . Upon recognition of a matching mRNA by RDE-1-bound siRNAs, RNA-dependent RNA Polymerases (RdRPs) are recruited, resulting in the production of numerous secondary siRNAs (20, 21) . Testing multiple target genes suggests that two different RdRPs are used for silencing: RRF-1 for genes expressed in somatic cells (20) (21) (22) and EGO-1 for genes expressed in the germline (23, 24) . Secondary siRNAs can bind the Argonaute NRDE-3 in somatic cells (25) or the Argonaute HRDE-1 in the germline (26) (27) (28) and subsequently accumulate within the nuclei of cells that express the target gene. Through these events, extracellular dsRNA can reduce the levels of mRNA and/or pre-mRNA of a target gene. While silencing by all extracellular dsRNA requires SID-1, DCR-1, and RDE-1, the requirement for other components can vary. For example, some genes expressed in somatic cells can be silenced by ingested dsRNA in the absence of RRF-1 (29) . …

Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegansGene. Published: 2001-01-01. 1,844 citations.

Gene knockdown in C. elegans can be induced by feeding RNAi [4], and the development of whole-genome feeding RNAi libraries means that RNAi can be used for large-scale genetic analysis, up to and including whole-genome screens [5–7]. Moreover, many of the cellular mechanisms that mediate feeding RNAi have been described. In particular, interfering RNA species enter cells using the dsRNA channel SID-1 [8–10], while within each cell the Argonaute protein RDE-1 is required to achieve gene knockdown [11]. … We fed these animals RNAi against GFP and found that, compared to control RNAi, GFP was efficiently knocked down in GABA neurons but was still present in other cells – including muscles, intestine, skin, and non-GABA neurons (Fig. 2A, B). This suggests that the RNAi response is limited to the tissue in which our artificial operon is expressed and does not spread to other adjacent cells or tissues. … The technique and strains presented here enable cell-specific knockdown in designated neurons simply by performing feeding RNAi.… However, all of these methods require the construction of a new transgenic animal for each gene of interest. The requirement for one strain per gene limits the usefulness of these techniques for questions involving many genes, because it is impractical to construct so many transgenic animals. Gene knockdown in C. elegans can be induced by feeding RNAi [4], and the development of whole-genome feeding RNAi libraries means that RNAi can be used for large-scale genetic analysis, up to and including whole-genome screens [5–7]. Moreover, many of the cellular mechanisms that mediate feeding RNAi have been described. In particular, interfering RNA species enter cells using the dsRNA channel SID-1 [8–10], while within each cell the Argonaute protein RDE-1 is required to achieve gene knockdown [11]. This molecular understanding has been used to develop a new method for mosaic gene expression that is generated by feeding RNAi, and thus is compatible with the study of many genes. For example, a muscle-specific rde-1 mosaic enables muscle specific knockdown in response to feeding RNAi [12]. … To determine the effectiveness of our approach, we expressed nuclear-localized GFP in all somatic cells of our GABA-specific RNAi strain and also marked the GABA neurons with mCherry. We fed these animals RNAi against GFP and found that, compared to control RNAi, GFP was efficiently knocked down in GABA neurons but was still present in other cells – including muscles, intestine, skin, and non-GABA neurons (Fig. 2A, B). This suggests that the RNAi response is limited to the tissue in which our artificial operon is expressed and does not spread to other adjacent cells or tissues. We also tested the viability of our strain when challenged with RNAi against an essential gene. ama-1 encodes the large subunit of C. elegans RNA polymerase II [23], and RNAi against ama-1 results in a larval arrest phenotype with penetrance approaching 100%. … Discussion The technique and strains presented here enable cell-specific knockdown in designated neurons simply by performing feeding RNAi. Our results in GABA, dopamine, glutamate, and acetyl choline-releasing neurons suggest that the technique can be used to limit feeding RNAi to any neuron or group of neurons. …

Lisa Timmons and Andy Fire [19] developed a technique for feeding worms bacteria expressing dsRNA and Tabara and Mello [20] showed that simply soaking worms in dsRNA is also effective in producing knock-down phenotypes. … The RNAi pathway started to be viewed as a two-step process consisting of siRNA production from long dsRNA and their subsequent utilization [1]. Studies in other systems showed that mature mRNA gets degraded in response to RNAi by an initial endonucleolytic cleavage event and subsequent mRNA destruction by exonuclease, suggesting a mechanism by which gene expression is reduced [1]2. Classical RNAi pathway The original studies of RNAi were performed by injecting dsRNA into the gonad or the body cavity of the worms and examining the phenotypes in the next generation [2]. Lisa Timmons and Andy Fire [19] developed a technique for feeding worms bacteria expressing dsRNA and Tabara and Mello [20] showed that simply soaking worms in dsRNA is also effective in producing knock-down phenotypes. These advances in introducing dsRNA allowed large-scale screens for RNAi deficient mutants [7] and also led to the production of genome wide collections of RNAi feeding constructs and genome-wide RNAi based screens [21]. The first mutants in the RNAi pathway identified by Tabara and Mello [7] were called RNAi deficient (rde). These original screens were aimed at identifying of viable mutants, resistant to RNAi targeting pos-1, a gene important for viability. … 1). Studies of PTGS in plants and molecular analysis of RNAi in vitro and in Drosophila tissue culture identified important intermediates in RNAi, short interfering RNAs (siRNAs), which most likely correspond to the heritable factor in C. elegans. Thus, the RNAi pathway started to be viewed as a two-step process consisting of siRNA production from long dsRNA and their subsequent utilization [1]. Studies in other systems showed that mature mRNA gets degraded in response to RNAi by an initial endonucleolytic cleavage event and subsequent mRNA destruction by exonuclease, suggesting a mechanism by which gene expression is reduced [1] (see review by Hammond in this issue). 1. Download: Download full-size image

In feeding RNAi, the intestinal lumen-localized transmembrane protein SID-2 is first required for endocytosis of ingested dsRNA into gut cells, and SID-1 is subsequently required for effective RNAi within intestinal cells likely to release dsRNA from endosomes (Winston et al. 2007; McEwan et al. 2012). … We found that only animals exposed to unc-22 food as adults efficiently produced twitching progeny (Figure 2B), even though the P0 animals fed on days 1 and 2 still exhibited a strong Unc-22 twitching phenotype immediately after RNAi exposure. … This result shows that rme-2 is not required in sid-1+ hermaphrodites for parental RNAi, even when the dsRNA is introduced by ingestion.sid-1-independent dsRNA transmission requires RME-2 The above results, showing sid-1 independent delivery of dsRNA from mothers to embryos and a sid-1-dependent step in the embryos for effective RNAi is similar to feeding RNAi, where the uptake and transport of ingested dsRNA is separable. In feeding RNAi, the intestinal lumen-localized transmembrane protein SID-2 is first required for endocytosis of ingested dsRNA into gut cells, and SID-1 is subsequently required for effective RNAi within intestinal cells likely to release dsRNA from endosomes (Winston et al. 2007; McEwan et al. 2012). A candidate receptor for dsRNA endocytosis in the germline is RME-2, an LDL receptor superfamily homolog that functions in oocytes as a yolk/lipoprotein receptor. In C. elegans, yolk is synthesized in the intestine, exported to the PC, and then taken up by oocytes via receptor-mediated endocytosis (Sharrock et al. … We split a batch of freshly hatched wild-type larvae into three populations, and exposed each to unc-22 RNAi food on either day 1 (L1/L2), day 2 (L3/L4), or day 3 (adult) after hatching, thoroughly washing off residual bacteria each day. After a final wash, animals were allowed to lay F1 progeny on non-RNAi food. We found that only animals exposed to unc-22 food as adults efficiently produced twitching progeny (Figure 2B), even though the P0 animals fed on days 1 and 2 still exhibited a strong Unc-22 twitching phenotype immediately after RNAi exposure. We then used these modified feeding RNAi conditions, which are effective for parental RNAi in wild-type animals, to determine whether rme-2 is required for parental RNAi. We found that the progeny of wild-type and rme-2 adults placed on unc-22 food show similar proportions of twitching progeny (Figure 2C). Note that rme-2 hermaphrodites are less fecund that wild-type hermaphrodites (Grant and Hirsh 1999). This result shows that rme-2 is not required in sid-1+ hermaphrodites for parental RNAi, even when the dsRNA is introduced by ingestion. Because inherited RNAi by dsRNA feeding requires adult exposure (Figure 2, B and C), it is reasonable to assume that the slightly slower development of rme-2 mutants differentially limited the period of adult exposure compared to wild-type animals when initially placed on the RNAi food as L4 larvae. …

Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. … In our research, we focus on body size regulation by the DBL-1/TGF-β pathway. The loss of function of the DBL-1 pathway results in small body size, including a shorter body length compared with wild-type animals7,10,11. Thus, screens for C. elegans body size mutants are capable of identifying TGF-β signaling components and modifiers. DBL-1 signaling is mediated by a conserved TGF-β signal transduction pathway that includes cell surface receptors and intracellular Smad signal transducers12. To test the effectiveness of RNAi by feeding for the identification of body size mutants, we used this technique to knock down DBL-1 pathway components: dbl-1/ligand; sma-2, sma-3, sma-4/Smads; and sma-6/receptor. … By using this protocol, knocking down DBL-1 pathway components showed expected body length phenotype. Those RNAi animals were about 68-95% in length compared with control animals. In previous studies, DBL-1 pathway loss of function genetic mutants in adulthood are about 50% shorter than wild-type animals7,10,11. Therefore, the RNAi feeding technique presented here did not fully eliminate gene activity.… Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- β - like ligand DBL-1, so this assay is appropriate for identification of TGF-β signaling components7. … ## Representative Results In our research, we focus on body size regulation by the DBL-1/TGF-β pathway. The loss of function of the DBL-1 pathway results in small body size, including a shorter body length compared with wild-type animals7,10,11. Thus, screens for C. elegans body size mutants are capable of identifying TGF-β signaling components and modifiers. DBL-1 signaling is mediated by a conserved TGF-β signal transduction pathway that includes cell surface receptors and intracellular Smad signal transducers12. To test the effectiveness of RNAi by feeding for the identification of body size mutants, we used this technique to knock down DBL-1 pathway components: dbl-1/ligand; sma-2, sma-3, sma-4/Smads; and sma-6/receptor. For our study, we used two different RNAi hypersensitive C. elegans strains to perform the experiment: eri-1;lin-15b and rrf-313,14. … Prior to initiating a large scale screen for other phenotypes, we would recommend a pilot screen with genes known to produce the phenotype of interest such as we describe here for dbl-1, sma-2, sma-3 sma-4, and sma-6. By using this protocol, knocking down DBL-1 pathway components showed expected body length phenotype. Those RNAi animals were about 68-95% in length compared with control animals. In previous studies, DBL-1 pathway loss of function genetic mutants in adulthood are about 50% shorter than wild-type animals7,10,11. Therefore, the RNAi feeding technique presented here did not fully eliminate gene activity. Thus, the method may be limited in its ability to identify pathway components that have a weak phenotype. Meanwhile, since there are many genes that regulate body length, genes identified from the screen also might not necessarily fall in DBL-1 pathway. …

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.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. Similar articles

Separately, pathogenic bacterial sRNAs are taken up by mothers and processed through the canonical RNAi pathway via Dicer activity in the intestine. Subsequent germline signaling involving the piRNA pathway and epigenetic modifiers 3 induce daf-7 expression in the ASI neuron, ultimately regulating avoidance of PA14. … Together, our data suggest that the ingestion of PA14 and subsequent uptake and Dicer-mediated processing of the PA14 ncRNA P11 in the intestine, followed by further processing and piRNA regulation in the germline, leads to downregulation of maco-1, which in turn is required for upregulation of daf-7 expression in the ASI and subsequent maternal avoidance behavior (Fig. 4g).… 2o). Our data support a model in which mothers respond acutely through the innate immune response 9 to pathogen exposure and metabolites, which induces daf-7 expression in the ASJ neuron 4, causing half of the avoidance behavior exhibited by trained worms (Fig. 2o inset; 1i, Extended Data Fig. 1j). Separately, pathogenic bacterial sRNAs are taken up by mothers and processed through the canonical RNAi pathway via Dicer activity in the intestine. Subsequent germline signaling involving the piRNA pathway and epigenetic modifiers 3 induce daf-7 expression in the ASI neuron, ultimately regulating avoidance of PA14. Only sRNAs are required for transgenerational inheritance of the avoidance behavior signal (Fig. 2n– o; 4g). Fig. 4: PA14 P11 sRNA induces avoidance through its target, maco-1. … Wild-type mothers exposed to E. coli expressing maco-1 dsRNA for 24hrs learned to avoid PA14 (Fig. 4f). Like E. coli-P11, this single exposure to maco-1 RNAi in the P0 generation was sufficient to recapitulate transgenerational memory of PA14 in naïve progeny. Therefore, maco-1 is specifically required for response to PA14 sRNAs, likely through its sequence matching P11. Together, our data suggest that the ingestion of PA14 and subsequent uptake and Dicer-mediated processing of the PA14 ncRNA P11 in the intestine, followed by further processing and piRNA regulation in the germline, leads to downregulation of maco-1, which in turn is required for upregulation of daf-7 expression in the ASI and subsequent maternal avoidance behavior (Fig. 4g). Discussion

In Caenorhabditis elegans, the model in which RNAi was initially uncovered, inhibition of gene expression can be accomplished by delivering double-stranded RNA into any part of the worm, since RNA is transported across cell boundaries in this organism. Thus RNAi can be accomplished in worms by feeding them bacteria expressing the dsRNA, soaking them in solutions containing dsRNA, or injecting them with RNA directly. … The worms fed with bacteria expressing dsRNA for bli-1 and gfp showed strong double RNAi phenotypes including a Bli phenotype and no GFP signal as the result of successful knock-down of both genes (Figure 1 B, Table S1). … In this study, we were able to accomplish the efficient knock-down of two genes simultaneously by a simple modification to the existing feeding RNAi strategy. Our results clearly showed that feeding RNAi with a DNA construct containing two or three gene fragments in one plasmid is efficient and reliable in knocking down the functions of the genes.Feeding RNAi RNAi is frequently used in reverse genetics studies in multiple cell types and organisms. In Caenorhabditis elegans, the model in which RNAi was initially uncovered, inhibition of gene expression can be accomplished by delivering double-stranded RNA into any part of the worm, since RNA is transported across cell boundaries in this organism. Thus RNAi can be accomplished in worms by feeding them bacteria expressing the dsRNA, soaking them in solutions containing dsRNA, or injecting them with RNA directly. Feeding is the preferred method for single gene RNAi since it is convenient and inexpensive. But knock-down of multiple genes using the feeding approaches can produce poor results; worms fed a mixture of two types of bacteria-each expressing dsRNA targeting a different gene-often show incomplete phenotypes. … In contrast, the knock-down efficiency of gfp was only slightly lower than that of single RNAi, suggesting that not all genes are sensitive to the dilution effect. To test the efficiency of feeding multiple genes in a single vector, we generated an RNAi construct containing both bli-1 and gfp in a single vector. The worms fed with bacteria expressing dsRNA for bli-1 and gfp showed strong double RNAi phenotypes including a Bli phenotype and no GFP signal as the result of successful knock-down of both genes (Figure 1 B, Table S1). Since only bli-1 RNAi showed the dilution effect, we examined whether either bli-1 or gfp showed the dilution effect when they were mixed with control bacteria containing the empty vector L4440. … The orders and orientations of the inserts did not affect the RNAi efficiency. With these data, we suggest that the new method is also suitable for endogenous genes, especially in cases where double feeding RNAi with two separate bacteria is ineffective for either one of the genes. In this study, we were able to accomplish the efficient knock-down of two genes simultaneously by a simple modification to the existing feeding RNAi strategy. Our results clearly showed that feeding RNAi with a DNA construct containing two or three gene fragments in one plasmid is efficient and reliable in knocking down the functions of the genes. The method described here may not be necessary for genes that show phenotypes by feeding two separate kinds of bacteria, but this method provides a good means of RNA interference for genes exhibiting reduced RNAi efficiency due to dsRNA dilution from bacteria expressing another dsRNA. While performing RNAi in a C. …

introduction of double-stranded RNA (dsRNA) results in the specific inactivation of an endogenous gene with corresponding sequence; this technique is known as RNA interference (RNAi) … RNAi can be performed by direct microinjection of dsRNA into adult hermaphrodite worms, by soaking worms in a solution of dsRNA, or by feeding worms Escherichia coli expressing target-gene dsRNAAbstract In Caenorhabditis elegans, introduction of double-stranded RNA (dsRNA) results in the specific inactivation of an endogenous gene with corresponding sequence; this technique is known as RNA interference (RNAi). It has previously been shown that RNAi can be performed by direct microinjection of dsRNA into adult hermaphrodite worms, by soaking worms in a solution of dsRNA, or by feeding worms Escherichia coli expressing target-gene dsRNA. We have developed a simple optimized protocol exploiting this third mode of dsRNA introduction, RNAi by feeding, which allows rapid and effective analysis of gene function in C. elegans. Furthermore, we have constructed a library of bacterial strains corresponding to roughly 86% of the estimated 19,000 predicted genes in C. elegans, and we have used it to perform genome-wide analyses of gene function. …

Unlike plants, C. elegans and Planaria can uptake dsRNA from the environment by feeding or soaking in a dsRNA solution. The exogenously supplied or microinjected dsRNA can trigger sequence‐specific gene silencing in the treated and adjacent tissues and silence the progeny of injected animals. … The movement of exogenous and transgenic dsRNA is well documented in C. elegans, it remains unknown whether endogenous sRNAs are transported in animals (see Box 1).Systemic RNAi in animals Although first identified in plants, systemic RNA silencing also occurs in diverse animals including worms (C. elegans; Fire et al, 1998), insects (Tribolium; Bucher et al, 2002) and Planaria (Newmark et al, 2003). Unlike plants, C. elegans and Planaria can uptake dsRNA from the environment by feeding or soaking in a dsRNA solution. The exogenously supplied or microinjected dsRNA can trigger sequence‐specific gene silencing in the treated and adjacent tissues and silence the progeny of injected animals. Thus, the long dsRNA or its derivatives must move across the cell membrane from cell‐to‐cell, unlike the situation in plants where the movement of the silencing signal occurs through a shared cytoplasm. This phenomenon implies the need for membrane‐bound transporters and, indeed, a transmembrane protein called SID‐1 has been identified in a forward genetic screen that is required for systemic RNA interference (RNAi) in C. elegans (Winston et al, 2002). … Since SID‐1 has higher affinity for longer dsRNA than sRNA duplexes (Feinberg and Hunter, 2003), it is highly likely that long dsRNA is the main component of the RNAi‐associated silencing signal in worms. Consistent with this idea, a mutation in RDE‐4, which prevents the processing of dsRNA into siRNA (Parrish and Fire, 2001), is not required for movement of a heritable silencing signal (Grishok et al, 2000). Although the movement of exogenous and transgenic dsRNA is well documented in C. elegans, it remains unknown whether endogenous sRNAs are transported in animals (see Box 1). Sources of dsRNA and siRNAs are present in the C. elegans genome (Ambros et al, 2003; Zhao et al, 2007) so it is likely that they are mobile. Consistent with this idea, SID‐1 imports synthetic pre‐miRNAs and long hairpin molecules, suggesting that it may also transport endogenous dsRNAs (Shih and Hunter, 2011).

In every experiment, injection of double-stranded RNA carrying a genetic code led to silencing of the gene containing that particular code. The protein encoded by that gene was no longer formed. … After a series of simple but elegant experiments, Fire and Mello deduced that double-stranded RNA can silence genes, that this RNA interference is specific for the gene whose code matches that of the injected RNA molecule, and that RNA interference can spread between cells and even be inherited.… Similar movements were seen in worms that completely lacked a functioning gene for the muscle protein. What had happened? When sense and antisense RNA molecules meet, they bind to each other and form double-stranded RNA. Could it be that such a double-stranded RNA molecule silences the gene carrying the same code as this particular RNA? Fire and Mello tested this hypothesis by injecting double-stranded RNA molecules containing the genetic codes for several other worm proteins. In every experiment, injection of double-stranded RNA carrying a genetic code led to silencing of the gene containing that particular code. The protein encoded by that gene was no longer formed. After a series of simple but elegant experiments, Fire and Mello deduced that double-stranded RNA can silence genes, that this RNA interference is specific for the gene whose code matches that of the injected RNA molecule, and that RNA interference can spread between cells and even be inherited. It was enough to inject tiny amounts of double-stranded RNA to achieve an effect, and Fire and Mello therefore proposed that RNA interference (now commonly abbreviated to RNAi) is a catalytic process. Fire and Mello published their findings in the journal Nature on February 19, 1998. Their discovery clarified many confusing and contradictory experimental observations and revealed a natural mechanism for controlling the flow of genetic information. …

double-stranded RNA was substantially more effective at producing interference than was either strand individually. After injection into adult animals, purified single strands had at most a modest effect, whereas double-stranded mixtures caused potent and specific interference. … Only a few molecules of injected double-stranded RNA were required per affected cell, arguing against stochiometric interference with endogenous mRNA and suggesting that there could be a catalytic or amplification component in the interference process.… Such effects have been proposed to result from a simple antisense mechanism that depends on hybridization between the injected RNA and endogenous messenger RNA transcripts. RNA interference has been used in the nematode Caenorhabditis elegans to manipulate gene expression 3, 4. Here we investigate the requirements for structure and delivery of the interfering RNA. To our surprise, we found that double-stranded RNA was substantially more effective at producing interference than was either strand individually. After injection into adult animals, purified single strands had at most a modest effect, whereas double-stranded mixtures caused potent and specific interference. The effects of this interference were evident in both the injected animals and their progeny. Only a few molecules of injected double-stranded RNA were required per affected cell, arguing against stochiometric interference with endogenous mRNA and suggesting that there could be a catalytic or amplification component in the interference process. Figure 1: Genes used to study RNA-mediated genetic interference in C.elegans.

Endogenous small interfering RNAs (endo-siRNAs), associated with ongoing silencing of protein-coding genes in normal worms, are produced by mechanisms that involve DCR-1 but, unlike miRNAs, also involve RDE-2, RDE-3, RDE-4, RRF-1, and RRF-3. … miRNAs and siRNAs function as guide RNAs, in conjunction with protein complexes containing members of the Argonaute family of RNA-binding proteins, to target complementary RNA transcripts for destruction or translational inhibitionAbstract C. elegans contains numerous small RNAs of ~21-24 nt in length. The microRNAs (miRNAs) are small noncoding RNAs produced by DCR-1-and ALG-dependent processing of self-complementary hairpin transcripts. Endogenous small interfering RNAs (endo-siRNAs), associated with ongoing silencing of protein-coding genes in normal worms, are produced by mechanisms that involve DCR-1 but, unlike miRNAs, also involve RDE-2, RDE-3, RDE-4, RRF-1, and RRF-3. The tiny noncoding (tncRNAs) are similar to endo-siRNAs in their biogenesis except that they are derived from noncoding sequences. These endo-siRNA-and tncRNA-based endogenous RNAi pathways involve some components, including DCR-1 and RDE-4, that are shared with exogenous RNAi, and some components, including RRF-3 and ERI-1, that are specific to endogenous RNAi. … RNAi can be elicited exogenously, by dsRNA supplied from outside the cell, or endogenously, from transcription of coding or noncoding genomic sequences (Ambros 2004; Baulcombe 2004; Lippman and Martienssen 2004; Mello and Conte 2004; Peragine et al. 2004; Allen et al. 2005; Grishok et al. 2005) . miRNAs and siRNAs function as guide RNAs, in conjunction with protein complexes containing members of the Argonaute family of RNA-binding proteins, to target complementary RNA transcripts for destruction or translational inhibition (Ambros 2004; Baulcombe 2004; Lippman and Martienssen 2004; Mello and Conte 2004) . In some cases, including Caenorhabditis elegans, distinct Argonaute proteins function in conjunction with siRNAs and microRNAs, respectively (Tabara et al. 1999; Grishok et al. 2001; Tijsterman et al. 2002b; Vastenhouw et al. 2003) . In other animals, the same Argonaute can function with both siRNAs and microRNAs (Williams and Rubin 2002; Meister et al. 2004; Okamura et al. 2004) . …

The phenomenon of RNA interference (RNAi), wherein introduction of dsRNA results in silencing of homologous genes, was first observed and reported in C. elegans. … In C. elegans extracts, DCR-1 dices long dsRNA into 23 bp duplexes (Ketting et al., 2001). Immuno-purified DCR-1 complexes digest dsRNA processively into siRNAs in the presence of ATP but terminate processing when ssRNA is encountered, demonstrating the specificity for dsRNA substrate (Ketting et al., 2001). … 26G RNAs repress target mRNA expression in their cognate cell types; loss of 26G RNAs does not result in ectopic expression within other cell types of the native male or female germline or inappropriate target expression in the opposite germline (Han et al., 2009).1. Introduction The phenomenon of RNA interference (RNAi), wherein introduction of dsRNA results in silencing of homologous genes, was first observed and reported in C. elegans. In addition, when the first small RNA, the lin-4 microRNA, was identified, it was thought to be a curiosity of C. elegans biology until the discovery of the conserved let-7 family of microRNAs. Since then, thousands of unique endogenous small RNAs have been found in C. … The dcr-1 mutant germline, however, shows an exogenous R NAi- de fective (Rde) phenotype and impaired transgene silencing, a related phenotype that reports on integrity of exo- and endo-RNAi pathways (Grishok et al., 2001; Ketting et al., 2001; Knight and Bass, 2001). In C. elegans extracts, DCR-1 dices long dsRNA into 23 bp duplexes (Ketting et al., 2001). Immuno-purified DCR-1 complexes digest dsRNA processively into siRNAs in the presence of ATP but terminate processing when ssRNA is encountered, demonstrating the specificity for dsRNA substrate (Ketting et al., 2001). Although 26G RNAs are transcribed by RRF-3, they do not show the signature 5’ triphosphate of unprimed synthesis by an RdRP (Makeyev and Bamford, 2002). … 26G RNAs repress target mRNA expression in their cognate cell types; loss of 26G RNAs does not result in ectopic expression within other cell types of the native male or female germline or inappropriate target expression in the opposite germline (Han et al., 2009). Table

RNAi can be elicited exogenously, by dsRNA supplied from outside the cell, or endogenously, from transcription of coding or noncoding genomic sequences (Ambros 2004; Baulcombe 2004; Lippman and Martienssen 2004; Mello and Conte 2004; Peragine et al. 2004; Allen et al. 2005; Grishok et al. 2005). … Animals mutant for the RdRp gene rrf-3, or the nuclease-encoding gene eri-1, exhibit an eri phenotype, in that they are more sensitive than wild type to gene inactivation by feeding or injection of double-stranded RNA (Simmer et al. 2002; Kennedy et al. 2004).… MicroRNAs and siRNAs are produced by the type III endonuclease Dicer from various forms of precursor double-stranded RNA (dsRNA), including short fold-back hairpin gene products (in the case of miRNAs), or, in the case of siRNAs, bidirectional transcripts, replicative intermediates from RNA-dependent RNA polymerase (RdRp) activity or possibly longer single-stranded transcripts with extensive secondary structure. RNAi can be elicited exogenously, by dsRNA supplied from outside the cell, or endogenously, from transcription of coding or noncoding genomic sequences (Ambros 2004; Baulcombe 2004; Lippman and Martienssen 2004; Mello and Conte 2004; Peragine et al. 2004; Allen et al. 2005; Grishok et al. 2005). miRNAs and siRNAs function as guide RNAs, in conjunction with protein complexes containing members of the Argonaute family of RNA-binding proteins, to target complementary RNA transcripts for destruction or translational inhibition (Ambros 2004; Baulcombe 2004; Lippman and Martienssen 2004; Mello and Conte 2004). In some cases, including Caenorhabditis elegans, distinct Argonaute proteins function in conjunction with siRNAs and microRNAs, respectively (Tabara et al. 1999; Grishok et al. … ### Compromising some endogenous RNAi pathways can enhance other RNAi pathways Animals mutant for the RdRp gene rrf-3, or the nuclease-encoding gene eri-1, exhibit an eri phenotype, in that they are more sensitive than wild type to gene inactivation by feeding or injection of double-stranded RNA (Simmer et al. 2002; Kennedy et al. 2004). The enhancement is not restricted only to exogenous RNAi, as rrf-3 and eri-1 mutants are also enhanced for the silencing of repetitive transgenes through RNAi pathways elicited without an external source of double-stranded RNA (Grishok et al. 2005; Robert et al. 2005). We observed that the accumulation of a specific siRNA or tncRNA can depend on the wild-type activity of rrf-3 or eri-1, and that numerous endogenous genes are up-regulated in these mutants. …

Interfering RNA is usually delivered to the nematode Caenorhabditis elegans by injection of double-stranded RNA (dsRNA) (Fire et al., 1998), orally by soaking nematodes in a concentrated solution of dsRNA (Tabara et al., 1998, Maeda et al., 2001) or by feeding worms bacteria engineered to produce dsRNA (Timmons and Fire, 1998).Introduction Interfering RNA is usually delivered to the nematode Caenorhabditis elegans by injection of double-stranded RNA (dsRNA) (Fire et al., 1998), orally by soaking nematodes in a concentrated solution of dsRNA (Tabara et al., 1998, Maeda et al., 2001) or by feeding worms bacteria engineered to produce dsRNA (Timmons and Fire, 1998). Microinjection of dsRNA is effective at inducing RNA interference (RNAi), but it is labour-intensive, requiring high levels of skill and is not well suited for large-scale screens. In contrast, oral delivery is quick and easy to perform. Indeed, many studies have used the feeding approach to screen nearly 78% of the 19,920 C. …

The enhancement is not restricted only to exogenous RNAi, as rrf-3 and eri-1 mutants are also enhanced for the silencing of repetitive transgenes through RNAi pathways elicited without an external source of double-stranded RNA (Grishok et al. 2005; Robert et al. 2005). … The finding that rrf-3 and eri-1 are involved in ongoing silencing of endogenous sequences supports the hypothesis that the enhancement of some RNAi pathways in these mutants could be a consequence of reduced activity of other endogenous RNAi pathways, resulting in elevation of critical components of the RNAi machinery.Compromising some endogenous RNAi pathways can enhance other RNAi pathways Animals mutant for the RdRp gene rrf-3, or the nuclease-encoding gene eri-1, exhibit an eri phenotype, in that they are more sensitive than wild type to gene inactivation by feeding or injection of double-stranded RNA (Simmer et al. 2002; Kennedy et al. 2004). The enhancement is not restricted only to exogenous RNAi, as rrf-3 and eri-1 mutants are also enhanced for the silencing of repetitive transgenes through RNAi pathways elicited without an external source of double-stranded RNA (Grishok et al. 2005; Robert et al. 2005). We observed that the accumulation of a specific siRNA or tncRNA can depend on the wild-type activity of rrf-3 or eri-1, and that numerous endogenous genes are up-regulated in these mutants. The finding that rrf-3 and eri-1 are involved in ongoing silencing of endogenous sequences supports the hypothesis that the enhancement of some RNAi pathways in these mutants could be a consequence of reduced activity of other endogenous RNAi pathways, resulting in elevation of critical components of the RNAi machinery. An elevation in RNAi components after loss of endogenous silencing could occur by numerous possible mechanisms. In one model, loss of endogenous silencing of mRNAs that encode limiting protein components of the core RNAi machinery could lead to increased levels of those proteins, analogous to the regulation of Dicer-Like-1 in Arabidopsis by a microRNA (Xie et al. 2003). …

Nineteen years after Lisa Timmons and Andy Fire first described RNA transfer from bacteria to C. elegans in an experimental settingCitation48 the biologic role of this trans-kingdom RNA-based communication remains unknown. Here we summarize our current understanding on the mechanism and potential role of such social RNA.Abstract Nineteen years after Lisa Timmons and Andy Fire first described RNA transfer from bacteria to C. elegans in an experimental settingCitation48 the biologic role of this trans-kingdom RNA-based communication remains unknown. Here we summarize our current understanding on the mechanism and potential role of such social RNA. Authors

Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegansNature. Published: 1998-02-01. 15,419 citations.