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Trends in Genetics,
1999]
Double -stranded RNA (dsRNA) has recently been shown to trigger sequence-specific gene silencing in a wide variety of organisms, including nematodes, plants, trypanosomes, fruit flies and planaria; meanwhile an as yet uncharacterized RNA trigger has been shown to induce DNA methylation in several different plant systems. In addition to providing a surprisingly effective set of tools to interfere selectively with gene function, these observations are spurring new inquiries to understand RNA-triggered genetic-control mechanisms and their biological roles.
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Methods Cell Biol,
1995]
DNA transformation assays in a whole organism provide experimental links between molecular structure and phenotype. Experiments with transgenic Caenorhabditis elegans start in general with the injection of DNA into the adult gonad. Effects on phenotype or gene expression patterns can be analyzed either in F1 progeny derived from the injected animals or in derived transgenic lines. Microinjection of C. elegans was first carried out by Kimble et al. (1982). Stinchcomb et al. (1985) then showed that injected DNA could be maintained for several generations in transgenic lines. The first selective methods for producing and maintaining transgenic lines were reported in 1986 (Fire, 1986). These methods have been considerably improved since then (Mello et al., 1991) , so that assays involving DNA transformation are now a standard part of the experimental repertoire for C. elegans.
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[
Annual Review of Genetics,
2000]
Mechanisms for repetition of DNA pose both opportunities and challenges to a functional genome: opportunities for increasing gene expression by amplification of useful sequences, and challenges of controlling amplification by unwanted sequences such as transposons and viruses. Experiments in numerous organisms have suggested the likely existence of a general mechanism for recognition of repeated character in DNA. This review focuses (a) on the nature of these recognition mechanisms, and (b) on types of chromatin modification and gene silencing that are used to control repeated DNA.
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Trends Genet,
1998]
Many fundamental natural processes have been uncovered not by preplanned scientific enquiry, but serendipitously by engineers and scientists who observed unexpected consequences of their manipulations. Biologists routinely use engineering to manipulate the expression of specific genes and, thus, understand (or benefit from) their function. Sometimes we wish to make a particular gene silent; at other times we want the genes to 'talk' more loudly.
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[
Methods Cell Biol,
1995]
In situ hybridization to RNA is an effective tool for the analysis of gene expression during development. This technique is particularly important for Caenorhabditis elegans, as isolation of RNA from specific tissues or developmental stages is generally not possible in this organism (see Chapter 20 in this volume). In addition, the availability of the complete cell lineage and the reproducibility of cell positions from one animal to the next allow RNA expression patterns to be analyzed at the level of individual cells. A number of in situ hybridization protocols have been developed for the detection of RNA in squashed, dissected, or sectioned tissues of C. elegans. More recently, protocols using whole-mount preparations of C. elegans have also been described. By preserving the three-dimensional structure of the specimen, whole-mount preparations facilitate the identification of specific cells and the analysis of complex expression patterns. In this chapter, we describe a protocol for detection of RNA in whole-mount C. elegans embryos. This procedure is based on protocols for Drosophila, which make use of highly sensitive digoxigenin-labeled probes...
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J Cell Sci Suppl,
1992]
The Caenorhabditis elegans protein, CeMyoD, is related to the vertebrate myogenic regulatory factors MyoD, myogenin, MRF-4 and Myf-5. Like its vertebrate counterparts, CeMyoD accumulates in the nucleus of striated muscle cells prior to the onset of terminal differentiation. CeMyoD also shares functional similarities with the vertebrate myogenic regulatory factors. Viral LTR driven expression of CeMyoD in mouse 10T1/2 cells can convert this cell line into myoblasts as well as efficiently trans-activate mouse muscle-specific promoters. Furthermore, mouse MyoD expression can activate a CeMyoD-beta-galactosidase reporter construct in a 10T1/2 co-transfection assay.
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Biotechnol J,
2008]
RNA interference (RNAi) is a mechanism displayed by most eukaryotic cells to rid themselves of foreign double-stranded RNA molecules. RNAi has now been demonstrated to function in mammalian cells to alter gene expression, and has been used as a means for genetic discovery as well as a possible strategy for genetic correction. RNAi was first described in animal cells by Fire and colleagues in the nematode, Caenorhabditis elegans. Knowledge of RNAi mechanism in mammalian cell in 2001 brought a storm in the field of drug discovery. During the past few years scientists all over the world are focusing on exploiting the therapeutic potential of RNAi for identifying a new class of therapeutics. The applications of RNAi in medicine are unlimited because all cells possess RNAi machinery and hence all genes can be potential targets for therapy. RNAi can be developed as an endogenous host defense mechanism against many infections and diseases. Several studies have demonstrated therapeutic benefits of small interfering RNAs and micro RNAs in animal models. This has led to the rapid advancement of the technique from research discovery to clinical trials.
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J Dent Res,
2008]
RNA interference (RNAi), an accurate and potent gene-silencing method, was first experimentally documented in 1998 in Caenorhabditis elegans by Fire et al., who subsequently were awarded the 2006 Nobel Prize in Physiology/Medicine. Subsequent RNAi studies have demonstrated the clinical potential of synthetic small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) in dental diseases, eye diseases, cancer, metabolic diseases, neurodegenerative disorders, and other illnesses. siRNAs are generally from 21 to 25 base-pairs (bp) in length and have sequence-homology-driven gene-knockdown capability. RNAi offers researchers an effortless tool for investigating biological systems by selectively silencing genes. Key technical aspects-such as optimization of selectivity, stability, in vivo delivery, efficacy, and safety-need to be investigated before RNAi can become a successful therapeutic strategy. Nevertheless, this area shows a huge potential for the pharmaceutical industry around the globe. Interestingly, recent studies have shown that the small RNA molecules, either indigenously produced as microRNAs (miRNAs) or exogenously administered synthetic dsRNAs, could effectively activate a particular gene in a sequence-specific manner instead of silencing it. This novel, but still uncharacterized, phenomenon has been termed ''RNA activation'' (RNAa). In this review, we analyze these research findings and discussed the in vivo applications of siRNAs, miRNAs, and shRNAs.
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[
1980]
Locomotory behavior has been investigated in a wide variety of animals, including nematodes. Many studies have concluded that locomotory behavior is under neurogenic control, i.e., during locomotion the coordinated contractions of relevant muscles results from a coordinated firing of motoneurons. Nematodes have long been known to have only a small number of neurons (about 250 in the adult female), an attribute which should make them an attractive simple system in which to examine the control of locomotion. It should be possible, for example, to examine the role of individual neurons in the control of locomotion and also to determine which interactions between neurons are responsible for locomotory behavior. These studies have not been previously attempted primarily because the anatomy of nematode motoneurons had not been determined (a situation recently remedied, see Section IV, B). In the absence of this knowledge, the analysis of nematode locomotion has concentrated on the role of interconnections between muscle cells, and has been dominated by the suggestion that the coordinated contraction of muscle cells results from these interconnections (i.e., myogenic control of locomotion). In these models, the nervous system is relegated to switching the musculature between different patterns of contraction (i.e., directions of wave propagation). This type of organization has been called neurocratic. It is not yet clear whether the control of locomotion in nematodes in neurogenic or myogenic. Our bias (which will be clear) is that it is the nervous system which plays the major role, but the crucial experiments
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Cell Biochem Biophys,
2006]
RNA interference (RNAi), through expression of small, double-stranded RNAs or short hairpin RNAs, produces sequence-specific mRNA degradation and decreased gene expression. Since its discovery in 1998 (Fire et al., 1998, Nature 391, 806-811), RNAi has rapidly become one of the most widely used technologies for exploring gene function in eukaryotic cells. Although the topic of RNAi has been the subject of a large number of excellent reviews, the focus of this article is on its application to the study of ion channel physiology in animal cells. In this regard, RNAi has provided definitive identification of ion channel subtypes responsible for both basal and stimulated ion conduction across the plasma membrane of several cell types. The approach has been particularly effective in identifying and establishing the contribution of auxiliary subunits and regulatory proteins to the overall function of ion channel complexes. Moreover, selective knockdown of ion channel expression has been a valuable means of demonstrating roles in the development of specific cell domains and in the normal growth of certain cell types. In this review, a brief description of the general mechanism of RNAi is presented, followed by a discussion of some important considerations for the in vitro application of this technology and in producing transgenic animals as models for human disease. We then describe several examples of where RNAi has been used to investigate the physiological role of ion channels in cells from model organisms (Caenorhabditis elegans and Drosophila melanogaster) and in mammalian cells.