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Nat Genet,
2003]
In the year that the Nobel Prize was awarded to Sydney Brenner, Bob Horvitz and Sir John Suslton, the 14th International C. elegans meeting was bound to be a celebration as well as a scientific meeting and social get-together. The celebratory mood reached its high point during the keynote address by Sydney Brenner, the 'Father of the Worm'. The address was classic Brenner, at once provocative and Delphic, with incisive analogies, witty anecdotes and sweeping dismissals (systems biology did not fare well), all delivered with his usual flair.
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Oncogene,
2004]
Cancer is a genetic disease that ultimately results from the failure of cells to respond correctly to diverse signals. Signal transduction and signal integration are highly complex, requiring the combinatorial interaction of multiple genes. Classical genetics in model organisms including Caenorhabditis elegans has been of immense use in identifying nonredundant components of conserved signalling pathways. However, it is likely that there is much functional redundancy in the informational processing machinery of metazoan cells; we therefore need to develop methods for uncovering such redundant functions in model organisms if we are to use them to understand complex gene interactions and oncogene cooperation. RNAi may provide a powerful tool to probe redundancy in informational networks. In this review, I set out some of the progress made so far by classical genetics in understanding redundancy in gene networks, and outline how RNAi may allow us to approach this problem more systematically in C. elegans. In particular, I discuss the use of genome-wide RNAi screens in C. elegans to identify synthetic lethal interactions and compare this with synthetic lethal interaction analysis in Saccharomyces cerevisiae.
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Cancer Metastasis Rev,
1999]
Genetic screens in the hermaphrodite nematode worm Caenorhabditis elegans resulted in the identification of the basal conserved machinery of apoptosis, arguably the single most important finding for our understanding of cell death. The last two years have seen enormous progress in the elucidation of the molecular interactions that lie at the heart of this conserved machinery, along with major insights both into how cell death is activated in the worm and into the mechanism of recognition and engulfment of the cell corpses. In this review, I set out the current models of cell death regulation and execution in C. elegans, focussing in particular on the similarities between cell death in C. elegans and vertebrates. Finally, I attempt to highlight key areas for future progress in cell death research in C. elegans and explore additional ways in which the worm can be used to understand the regulation of cell death in mammals.
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[
West Coast Worm Meeting,
2000]
RNAi is a powerful and specific means of inhibiting gene function. However, the most widely used technique for RNAi, the injection of dsRNA into adult animals, is too labour-intensive to allow efficient genome-wide screening for gene function by RNAi. An alternative approach for RNAi is to feed bacteria expressing dsRNA to worms. We have determined conditions for which RNAi by bacterial feeding is as potent as RNAi by injection. We have constructed a library of dsRNA-expressing bacteria that can be used to target 90% of genes on chromosome I by RNAi. This reagent can be used for an unlimited number of low-cost screens for gene function. We have used this library to screen for all genes on chromosome I that give a clear phenotype when targeted by RNAi. We will present our results and discuss their implications for genome-wide functional analysis of C. elegans genes.
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[
International Worm Meeting,
2003]
C. elegans is in many ways an ideal organism in which to examine signal transduction. The genome encodes components of almost all known pathways and these genes are organised in well-conserved modules. Furthermore, unlike many other metazoa, the output of signaling in the worm is mainly non-stochastic i.e. given a certain combination of signals in a particular cell, the developmental decision is always the same.In an ideal world, we would like to know all the components of all pathways and their ordering that is, the way that information flows and is processed in a pathway, and the ways that pathways overlap and thus integrate information. This information should ultimately lead to an explanation for signaling specificity and the amazing balance in most pathways between sensitivity to low signal input coupled to great robustness. We present a combined strategy to map out signaling pathways in the worm, including genome-wide RNAi screens, high-throughput yeast two-hybrid analysis and bioinformatic approaches. Each approach is independent and systematic, and combining them should allow relatively (!) unbiased assembly of signaling pathways. As well as these abstract fuzzy notions, we also present real unpublished data arising from firstly RNAi screens examining modulators of ras signaling in the vulva and secondly yeast two-hybrid screens mapping out a physical interaction space of signaling.
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[
International C. elegans Meeting,
2001]
We know the complete genome sequence of C.elegans , and can therefore predict the sequences of nearly all encoded genes. A major problem remains to understand what roles each gene plays in the development and function of the worm. The approach that we are taking to this problem is to use RNA-mediated interference (RNAi) to inhibit the function of each gene in the genome. To do this, we are constructing libraries of dsRNA-expressing bacteria; each strain targets a single predicted gene by RNAi when fed to worms. This feeding technique, pioneered by Timmons and Fire, allows for high throughput RNAi screening and, furthermore, the libraries can be used for an unlimited number of future screens (for example, to identify genes with subtle or conditional phenotypes). We previously published the results of screening 87% of chromosome I genes (Fraser et al. 2000). We have now screened 88% of chromosome II genes and are currently screening an X chromosome library we have constructed. Data from chromosomes I and II are similar, with phenotypes detected for 14% of chr I and 12% of chr II genes. Also, as was seen on chromosome I, we find that fewer genes have an RNAi phenotype in the duplicated regions of chromosome II. We present an update of the project including a comparison of results obtained from chromosomes I, II, and X. In addition to identifying biological roles for many genes, we can also use our data to infer models of genome evolution and to discern relationships between the types of genes involved in different developmental processes. Finally, to increase our ability to identify RNAi phenotypes, we are screening for mutants with an increased sensitivity to RNAi; these should prove valuable for future RNAi-based screens.
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European Worm Meeting,
2000]
We have begun a reverse genetic screen of the C. elegans genome using RNA-mediated interference to identify a majority of genes involved in spindle orientation and early embryonic polarity. Because RNAi by injection is both time consuming and very expensive, we decided to perform the screen using RNAi by feeding. We first optimised feeding conditions using a test set of 20 maternal-effect embryonic lethal genes, and we have shown that we can detect these genes by feeding as reliably as we can by injection. In addition, performing the screen with RNAi by feeding allows us to detect many post-embryonic phenotypes which we do not see by injection. We are employing the following methodology for our screen: We amplify each predicted gene using primer pairs obtained from Research Genetics, and then TA clone them into the L4440 feeding vector (Timmons and Fire, 1998). These ligation products are transformed into the HT115 RNase- E. coli strain, and the resulting colonies are screened for the appropriate insert. We can successfully clone approximately 90% of all predicted genes using this method. Bacteria with correct inserts are subsequently seeded onto NGM plates with IPTG and Amp and are also frozen into a glycerol stock, thus resulting in a feeding library useful for future screens or for repeating feeding of individual genes. L4-stage hermaphrodites are fed bacteria expressing dsRNA corresponding to each predicted gene and are then transferred individually to wells, allowed to lay eggs, and removed. The embryos are then scored for lethality; those that hatch can be scored for adult phenotypes. For genes that give an embryonic lethal phenotype, dsRNA is made and injected to confirm the feeding phenotype and also to make 4D recordings of the early cell divisions. Currently, about 10% of genes have been found to give an embryonic lethal phenotype, and a further 10% have given post-embryonic phenotypes. Using this approach, by the time of the meeting, we hope to have screened most of the predicted genes from chromosome I. We will present preliminary data from our screen.
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[
East Coast Worm Meeting,
2000]
C. elegans is an ideal model system for the dissection of diverse biological processes using reverse genetics. Firstly, the entire sequence of the genome is known and is predicted to contain ~19,000 genes; secondly, RNA-mediated inhibition allows the directed and specific inhibition of individual genes. RNAi is thus an ideal tool for a genome-wide analysis of gene function in C. elegans. However, the most widely used methods for RNAi in C. elegans require the sythesis of RNA in vitro; these methods are labour intensive, high cost and ultimately result in limited amounts of dsRNA, thus precluding multiple subsequent screens. It has previously been shown that RNAi can be carried out by feeding bacteria that express dsRNA to adult worms. We have determined conditions for which feeding dsRNA-expressing bacteria to worms results in potent RNAi effects; once constructed, such bacterial strains can be used for unlimited experiments at minimal cost. We present here the construction and screening of a bacterially-expressed dsRNA library spanning the entire of Chromosome I, which contains ~17% of all C. elegans genes. We will describe our results for the screen so far. This is the first attempt at a comprehensive reverse-genetic analysis in any eukaryote and marks a significant advance in functional genomics.
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International Worm Meeting,
2011]
RNAi screens have revolutionized genetic screens in the worm. Like any genetic screen, an RNAi screen relies completely on phenotyping accuracy. However, to date there have been no published genome-scale screens using automated quantitative phenotyping - all phenotyping has been manual and at best semi-quantitative. Furthermore, although over 50 distinct phenotypes have been examined at genome-scale, the single most important phenotype for any evolutionary studies - fitness - has been completely ignored. We present here three complementary quantitative methods to direct assess the effect of RNAi on fitness and provide strong evidence that these methods are more sensitive than any manual screening as well as yielding highly reproducible quantitative measures of phenotypic strength. We illustrate how we have applied these methods to the study of natural variation in C. elegans and demonstrate the critical importance of quantitation to identify subtle defects and to correct for strain to strain growth differences.
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[
International Worm Meeting,
2005]
Complex genetic interactions underlie much of biology and are at the root of many human diseases. However, most frequently, our analysis in model organisms of the genetic basis for phenotypic traits involves the perturbation of single genes, rather than the systematic examination of gene interactions. We are attempting to explore genetic interactions more systematically using screens for synthetic lethals. A synthetic lethal interaction between two genes, A and B, occurs if deleting either A or B yields a viable organism, whereas removing both A and B is lethal. In yeast, systematic screens for pairs of synthetic lethal genes have uncovered a large amount about the wiring of basic cellular biology. We are identifying synthetic lethals in C. elegans by comparing the sets of lethal genes generated by RNAi in wild-type and in mutant backgrounds. To screen sufficient numbers of genes, we have developed an efficient high-throughput method for inducing RNAi by feeding and also for the automated microscopic analysis of the resulting phenotypes. We induce RNAi by feeding dsRNA-expressing bacteria to worms growing in liquid culture this can be done in 96-well format and is as efficient as other ways of RNAi by feeding. To analyse phenotypes, we have set up an automated image analysis system which we will describe this generates precise quantitative measurements of sterile and lethal phenotypes. These RNAi feeding and analysis protocols allow us to analyse ~1200 genes targeted by RNAi per day. We present the results of screens for genes that are synthetic lethal with
efl-1/EF2 gene; this gene is part of the
lin-35/Retinoblastoma complex that is involved in regulation of the cell cycle regulation and of vulval development. We find multiple novel biological connections including
ncl-1 and a component of SWI/SNF and with SynMuv genes. We will present these data and discuss our results.