Book contents
- Frontmatter
- Contents
- Foreword by Andrew Fire
- Foreword by Marshall Nirenberg
- List of Contributors
- Introduction
- Section one Basic RNAi, siRNA, microRNAs and gene-silencing mechanisms
- Section two Design, synthesis of siRNAs
- Section three Vector development and in vivo, in vitro and in ovo delivery methods
- Section four Gene silencing in model organisms
- Section five Drug target validation
- Section six Therapeutic and drug development
- Section seven High-throughput genome-wide RNAi analysis
- 30 High-throughput RNAi by soaking in Caenorhabtis elegans
- 31 Tools for integrative genomics: Genome-wide RNAi and expression profiling in Drosophila
- 32 Microarray analysis and RNA silencing to determine genes functionally important in mesothelioma
- 33 High-throughput RNA interference
- 34 Generation of highly specific vector-based shRNAi libraries directed against the entire human genome
- Index
- Plate section
- References
30 - High-throughput RNAi by soaking in Caenorhabtis elegans
Published online by Cambridge University Press: 31 July 2009
Book contents
- Frontmatter
- Contents
- Foreword by Andrew Fire
- Foreword by Marshall Nirenberg
- List of Contributors
- Introduction
- Section one Basic RNAi, siRNA, microRNAs and gene-silencing mechanisms
- Section two Design, synthesis of siRNAs
- Section three Vector development and in vivo, in vitro and in ovo delivery methods
- Section four Gene silencing in model organisms
- Section five Drug target validation
- Section six Therapeutic and drug development
- Section seven High-throughput genome-wide RNAi analysis
- 30 High-throughput RNAi by soaking in Caenorhabtis elegans
- 31 Tools for integrative genomics: Genome-wide RNAi and expression profiling in Drosophila
- 32 Microarray analysis and RNA silencing to determine genes functionally important in mesothelioma
- 33 High-throughput RNA interference
- 34 Generation of highly specific vector-based shRNAi libraries directed against the entire human genome
- Index
- Plate section
- References
Summary
Introduction
The phenomenon of RNA-mediated interference (RNAi) was first discovered in the nematode Caenorhabditis elegans (Fire et al., 1998), in which introduction of double-stranded RNA (dsRNA) causes specific inactivation of genes with corresponding sequences. RNAi was soon recognized as an experimentally simple and technically undemanding method for gene knockdown in this organism. The emergence of this new technology, which coincided with the completion of the sequencing of the C. elegans genome (The C. elegans Sequencing Consortium, 1998), has brought about a dramatic shift in the experimental strategies adopted in the study of this organism, and greatly expanded our understanding of gene function at the whole-genome level. Our group have established an optimized “soaking method” for dsRNA delivery into worms (Maeda et al., 2001), and applied this technique for large-scale analysis of gene function. In this paper, I describe the characteristics of the “soaking method,” and its application in the study of developmental processes in C. elegans.
RNAi in C. elegans
After the finding of RNAi in C. elegans, a series of subsequent analyses demonstrated that wide range of animals, plants, and fungi has RNAi-related gene regulatory mechanisms (Cogoni and Macino, 2000). Biochemical and genetic approaches, conducted mainly in C. elegans and Drosophila, have revealed the evolutionarily conserved nature of the molecular mechanism of RNAi (see other chapters in this book for detail). Although the “core RNAi mechanism” appears to be conserved among diverse organisms, some aspects of RNAi observed in C. elegans are not universally found in other species.
- Type
- Chapter
- Information
- RNA Interference TechnologyFrom Basic Science to Drug Development, pp. 419 - 432Publisher: Cambridge University PressPrint publication year: 2005
References
and (1987). glp-1 is required in the germ line for regulation of the decision between mitosis and meiosis in C. elegans. Cell, 51, 589–599CrossRefGoogle ScholarPubMed
and (1989). Transcript analysis of glp-1 and lin-12, homologous genes required for cell interactions during development of C. elegans. Cell, 58, 565–571CrossRefGoogle ScholarPubMed
and (2000). Post-transcriptional gene silencing across kingdoms. Current Opinions in Genetics and Development, 10, 638–643CrossRefGoogle ScholarPubMed
, , , , , , , and (2002). Rates of behavior and aging specified by mitochondrial function during development. Science, 298, 2398–2401CrossRefGoogle ScholarPubMed
, , , , and (2001). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 411, 494–498CrossRefGoogle ScholarPubMed
, , , , and (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 391, 806–811CrossRefGoogle ScholarPubMed
, , , , and (2000). Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature, 408, 325–330CrossRefGoogle ScholarPubMed
, , , , , , , , , , et al. (2000). Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III. Nature, 408, 331–336CrossRefGoogle Scholar
, , , and (2000). Genomic analysis of gene expression in C. elegans. Science, 290, 809–812CrossRefGoogle ScholarPubMed
and (2003). Genome-wide RNAi screening in Caenorhabditis elegans. Methods, 30, 313–321CrossRefGoogle ScholarPubMed
, , , , , , , , , , et al. (2003). Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature, 421, 231–237CrossRefGoogle ScholarPubMed
, , , , and (2001). Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans. Genome Biology, 2, RESEARCH0002Google ScholarPubMed
, , , , , and (2002). X-chromosome silencing in the germline of C. elegans. Development, 129, 479–492Google ScholarPubMed
, , , , , , , and (2001). A gene expression map for Caenorhabditis elegans. Science, 293, 2087–2092CrossRefGoogle ScholarPubMed
, , , , and (2000). SPK-1, a C. elegans SR protein kinase homologue, is essential for embryogenesis and required for germline development. Mechanisms of Development, 99, 51–64CrossRefGoogle Scholar
, , , , and (2003). A systematic RNAi screen identifies a critical role for mitochondria in C. elegans longevity. Nature Genetics, 33, 40–48CrossRefGoogle ScholarPubMed
, , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and (2004). A Map of the Interactome Network of the Metazoan C. elegans. Science, 303, 540–543CrossRefGoogle ScholarPubMed
, , and (2001). Large-scale analysis of gene function in Caenorhabditis elegans by high-throughput RNAi. Current Biology, 11, 171–176CrossRefGoogle ScholarPubMed
, and (1998). RNA as a target of double-stranded RNA-mediated genetic interference in Caenorhabditis elegans. Proceedings of the National Academy of Sciences USA, 95, 15502–15507CrossRefGoogle ScholarPubMed
, , , and (2002). Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes & Development, 16, 948–958CrossRefGoogle ScholarPubMed
, , , and (2000). RNAi analysis of genes expressed in the ovary of Caenorhabditis elegans. Current Biology, 10, 1619–1622CrossRefGoogle ScholarPubMed
, , , , , and (2002). Gene clustering based on RNAi phenotypes of ovary-enriched genes in C. elegans. Current Biology, 12, 1959–1964CrossRefGoogle ScholarPubMed
, and (1987). The glp-1 locus and cellular interactions in early C. elegans embryos. Cell, 51, 601–611CrossRefGoogle ScholarPubMed
, , , , , , , , , , et al. (2003). C. elegans ORFeome version 1.1: Experimental verification of the genome annotation and resource for proteome-scale protein expression. Nature Genetics, 34, 35–41CrossRefGoogle ScholarPubMed
, , , , , , , , , , et al. (2001). Open-reading-frame sequence tags (OSTs) support the existence of at least 17300 genes in C. elegans. Nature Genetics, 27, 332–336CrossRefGoogle ScholarPubMed
, , , , , , , , , and (2000). A global profile of germline gene expression in C. elegans. Molecular Cell, 6, 605–616CrossRefGoogle ScholarPubMed
, , , , , , and (2001). On the role of RNA amplification in dsRNA-triggered gene silencing. Cell, 107, 465–476CrossRefGoogle ScholarPubMed
, , , , , , , and (2003). Genome-wide RNAi of C. elegans using the hypersensitive rrf-3 strain reveals novel gene functions. Public Library of Science Biology, 1, E12Google ScholarPubMed
, , , , , , and (2002). Loss of the putative RNA-directed RNA polymerase RRF-3 makes C. elegans hypersensitive to RNAi. Current Biology, 12, 1317–1319CrossRefGoogle ScholarPubMed
, , , and (2001). Worm Base: network access to the genome and biology of Caenorhabditis elegans. Nucleic Acids Research, 29, 82–86CrossRefGoogle Scholar
, and (1998). RNAi in C. elegans: Soaking in the genome sequence. Science, 282, 430–431CrossRefGoogle Scholar
, , , and (2000). Heritable and inducible genetic interference by double-stranded RNA encoded by transgenes. Nature Genetics, 24, 180–183CrossRefGoogle ScholarPubMed
The C. elegans Sequencing Consortium. (1998). Genome sequence of the nematode C. elegans: A platform for investigating biology. Science, 282, 2012–2018CrossRef
, and (2002). Systemic RNAi in C. elegans requires the putative transmembrane protein SID-1. Science, 295, 2456–2459CrossRefGoogle Scholar
- 3
- Cited by