Research Update
TRENDS in Genetics Vol.18 No.1 January 2002
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Techniques & Applications
RNA interference by feeding in Paramecium Angélique Galvani and Linda Sperling RNA-mediated interference (RNAi) of gene expression is based on the ability of experimentally introduced doublestranded RNA (dsRNA) to trigger degradation of homologous RNAs in a cell or organism. Conserved across eukaryotic phyla, this phenomenon is closely related to post-transcriptional gene silencing (PTGS), whereby expression of transforming DNA can lead to activation of the same dsRNAbased degradation mechanism. In the ciliate Paramecium, PTGS is routinely used for functional analysis. We report here that the tools developed for delivering dsRNA to nematode worms by ingestion of engineered bacteria are easily adapted to Paramecium,
allowing potent and rapid genetic interference suitable for large-scale screens. PTGS can be induced in Paramecium by transformation of the somatic nucleus with transgenes critically lacking a 3′ untranslated region (UTR) [1,2], leading to production of dsRNA [2], which triggers the RNA degradation mechanism common to RNAi and PTGS [3]. Direct injection of dsRNA in Paramecium results in transitory genetic interference from which cells recover within 48 hours. The transitory nature of RNAi in Paramecium could be due to dilution of the injected molecules, because the cells undergo eight to ten divisions during this time.
Complete gene coding regions were inserted into the L4440 vector between the double T7 promoter. The different genes tested, their function, the phenotype observed and the observation methods are indicated in Table I. For each gene, phenotypes of cells from at least ten independent clones were screened after 48 hours of incubation in dsRNA-expressing bacteria. TMP4a-silenced cells and ND7-silenced cells were screened on their ability to secrete exocytotic granules. The picric acid test was used to evaluate exocytotic capacity; this lethal fixative provokes secretion of all exocytosis-competent granules that are easily visualized around the cell by low magnification dark-field microscopy. Phase contrast microscopy at high magnification allowed observation of the shape and intracellular localization of the granules within exocytosis-deficient cells. The infraciliary lattice (ICL) is a continuous contractile cytoskeletal network that underlies the whole cell cortex, composed of Paramecium centrins. Its disassembly in cells fed with ICL1a dsRNA-expressing bacteria was examined by immunocytochemistry using an antibody directed against ICL1 polypeptides [a]. For all genes tested, the phenotypes
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Genetic interference by injection of dsRNA was first reported in the nematode Caenorhabditis elegans [4], and Fire and colleagues demonstrated that feeding C. elegans with bacteria engineered to express dsRNA can induce RNAi phenotypes as efficiently as direct injection [5–7]. Because standard laboratory conditions for Paramecium growth involve feeding with enterobacteria [8], which enter a cycle of phagocytosis [9], we decided to see whether feeding could be used for continuous delivery of dsRNA. Using the double-T7-promoter feeding vector L4440 [5] and an RNase III-deficient feeding strain of E. coli with an IPTGinducible T7 polymerase, HT115(DE3) [7],
obtained by feeding were equivalent to those observed in PTGS studies. After 24 hours of feeding (with the exception of the NSF gene, 100% lethal after 24 hours), clones were heterogeneous, but strongly affected: 60–80% of the cells were completely silenced, whereas the others presented hypomorphic phenotypes. No
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Research Update
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we studied the effects of feeding using genes that have well-characterized PTGS phenotypes. Results and discussion
As shown in Box 1, we obtained the same, complete loss-of-function phenotypes for each of the genes tested as that previously observed for the same genes using PTGS, whereas parallel control experiments with uninduced bacteria showed normal cell growth and unaltered phenotypes. We verified that the loss-offunction phenotypes are mediated by dsRNA. Feeding experiments with bacteria expressing only sense or antisense RNA for a given gene led to no detectable phenotype (data not shown). We also found that the effects of RNAi are rapidly and completely reversed upon returning the cells to medium containing the usual, non-engineered bacterial strain. RNAi feeding technology is thus a potent, rapid and reliable means to study diverse gene functions in Paramecium. Compared with PTGS, which relies on transformation by injection of DNA, feeding has decided advantages. It is far less labour-intensive, it can be scaled up for biochemical studies even of essential genes, and it can be used to study not
TRENDS in Genetics Vol.18 No.1 January 2002
only the effects of turning a gene off, but also recovery when the gene is turned back on. The approach is well suited to large-scale screening [10] and should provide an invaluable tool for Paramecium functional genomics, in complement to the developing sequence project [11]. Perhaps most exciting, the success of RNAi by feeding in Paramecium raises the possibility of its use for any organism that can feed by phagocytosis of bacteria, including ciliates such as Tetrahymena, amoebae such as Acanthamoeba, Dictyostelium and the pathogen Entamoeba. Acknowledgements
We are indebted to Renaud Legouis for technical advice and encouragement, and to Andrew Fire for reagents. We thank Janine Beisson, Jean Cohen and Marine Froissard for unpublished data and useful discussions. A. Galvani was supported by a fellowship from the Association pour la Recherche sur le Cancer. References 1 Ruiz, F. et al. (1998) Homology-dependent gene silencing in Paramecium. Mol. Biol. Cell 9, 931–943 2 Galvani, A. and Sperling, L. (2001) Transgenemediated post-transcriptional gene silencing is inhibited by 3′ non-coding sequences in Paramecium. Nucleic Acids Res. 29, 4387–4394
3 Matzke, M. et al. (2001) RNA: guiding gene silencing. Science 293, 1080–1083 4 Fire, A. et al. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811 5 Timmons, L. and Fire, A. (1998) Specific interference by ingested dsRNA. Nature 395, 854 6 Kamath, R.S. et al. (2000) Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans. Genome Biol. research0002.1-0002.10 (http://genomebiology.com) 7 Timmons, L. et al. (2001) Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans. Gene 263, 103–112 8 Sonneborn, T.M. (1970) Methods in Paramecium research. Meth. Cell Physiol. 4, 241–339 9 Fok, A.K. and Allen, R.D. (1990) The phagosome–lysosome membrane system and its regulation in Paramecium. Int. Rev. Cytol. 123, 61–94 10 Fraser, A.G. et al. (2000) Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature 408, 325–330 11 Dessen, P. et al. (2001) Paramecium genome survey: a pilot project. Trends Genet. 17, 306–308
Angélique Galvani Linda Sperling* Centre de Génétique Moléculaire, CNRS, 91198 Gif-sur-Yvette, France. *e-mail:
[email protected]
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