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[
Methods Mol Biol,
1999]
The use of antibodies to visualize the distribution and subcellular localization of gene products powerfully complements genetic and molecular analysis of gene function in Caenorhabditis elegans. Double and triple staining protocols are particularly useful for several reasons. First, colonization of proteins either within tissues or at a subcellular level can be examined. Second, costaining with stage-specific or tissue-specific markers can define the timing and tissue specificity of antigen expression. For these types of studies it is useful to be able to collect data from multiple fluorescence wavelengths simultaneously. A confocal microscope equipped with a krypton/argon laser can simultaneously detect up to three different antigens. Using a confocal microscope it is also possible to collect a series of optical sections through a sample that allows observation of changes in distribution of the antigen in different focal planes of the tissue or cell.
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[
Methods Mol Biol,
2000]
The complete description of its nearly invariant cell lineage and the growing availability of cloned genes and markers for the cell lineage make Caenorhabditis elegans particularly favorable for mosaic analysis, and the literature is rich in examples that prove the usefulness of this approach. Because genetic mosaic analysis in C. elegans has recently been reviewed by Herman who developed many of the techniques, this review will be more concerned with the recent technical advances rather than with an extensive background of the approach. First we shall present a brief summary of the principles of mosaic analysis as it is typically performed in C. elegans. This will be followed by a description of markers that indicate mosaicism and by a discussion of a hypothetical analysis of an essential gene.
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[
Methods Mol Biol,
2000]
Complete or partial embryonic cell lineages are available for several animal model systems. In the case of the nematode Caenorhabditis elegans, the entire embryonic cell lineage has been determined and is largely invariant. This makes cell lineage analysis a potentially useful tool for assessing mutant phenotypes in C. elegans. Indeed, lineage analysis of some mutants has shown that one cell can be transformed into a different cell resulting in duplication or absence of certain tissues...
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[
Methods Mol Biol,
1999]
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[
Methods Mol Biol,
1999]
The nematode Caenorhabditis elegans has gained widespread popularity for use in addressing many biological problems, particularly those relating to development (for brief topical reviews, see 1-5; for comprehensive treatises, see 6-10). This can be attributed to both inherent properties of the organism as well as the collegiality extant within the "worm community". With respect to the former, C. elegans is extremely east to grow in the laboratory (animals are typically propagated on agar-filled Petri dishes seeded with the bacterium Escherichia coli) and possesses a short generation time (3 d at 20C). The system is genetically robust, with the availability of thousands of mutants as well as the existence of a physical map whose sequencing (over 82 Mb finished at present) is scheduled for completion in 1999. Developmental studies have been advantaged by the animal's transparent nature, facilitating complete elucidation of C. elegans' largely invariant cell
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Methods Mol Biol,
2006]
The identification and cloning of the green fluorescent protein (GFP) from jellyfish marks the beginning of a new era of fluorescent reporters. In Caenorhabditis elegans, genetically encoded markers like the fluorescent proteins of the GFP family became the reporter of choice for gene expression studies and protein localization. The small size and transparency of the worm allows the visualization of in vivo dynamics, which increases the number of potential applications for fluorescent reporters tremendously. In combination with subcellular tags, GFP can be used to label subcellular structures like synapses allowing novel approaches to study developmental processes like synapse formation. Other fluorescent labels like small organic dyes, which are in widespread use in cell culture systems, are rarely used in C. elegans owing to difficulties in applying these labels through the impenetrable cuticle or eggshell of the animal. A notable exception is the use of lipophilic dyes, which are taken up by certain sensory neurons in the intact animal and can be introduced into the embryo after puncturing of the egg shell. This chapter covers the use of fluorescent dyes and fluorescent proteins in C. elegans. Emphasis is placed on microscopic techniques including wide field and confocal microscopy as well as time-lapse recordings. The use of fluorescent proteins as transgenic markers and image processing of fluorescence images are briefly discussed.
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[
Methods Cell Biol,
1995]
This chapter has two aims. First, we describe one method, the electropharyngeogram (EPG), insufficient detail that a Caenorhabditis elegans researcher unfamiliar with electrophysiological methods could set up the apparatus and get useful results. Second, we describe more generally for researchers familiar with electrophysiological methods how they may be applied to C. elegans. We do not describe methods for electrophysiological investigation of C. elegans sperm.
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[
Methods Cell Biol,
1995]
Sequence analysis of cosmids from C. elegans and other organisms currently is best done using the random or "shotgun" strategy (Wilson et al., 1994). After shearing by sonication, DNA is used to prepare M13 subclone libraries which provide good coverage and high-quality sequence data. The subclones are assembled and the data edited using software tools developed especially for C. elegans genomic sequencing. These same tools facilitate much of the subsequent work to complete both strands of the sequence and resolve any remaining ambiguities. Analysis of the finished sequence is then accomplished using several additional computer tools including Genefinder and ACeDB. Taken together, these methods and tools provide a powerful means for genome analysis in the nematode.
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[
Methods Cell Biol,
1995]
The nematode Caenorhabditis elegans is a small, rapidly growing organism that can easily be raised in the laboratory on the bacterium Escherichia coli. Because C. elegans is a self-fertilizing hermaphrodite, it is possible to readily grow large quantities of the organism in swirling liquid cultures and also possible to propagate severely incapacitated mutants. The rapidity of growth and the ability to self-fertilize necessitate special measures to establish a synchronous culture.
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[
Methods,
2015]
For a long time, autophagy has been mainly studied in yeast or mammalian cell lines, and assays for analyzing autophagy in these models have been well described. More recently, the involvement of autophagy in various physiological functions has been investigated in multicellular organisms. Modification of autophagy flux is involved in developmental processes, resistance to stress conditions, aging, cell death and multiple pathologies. So, the use of animal models is essential to understand these processes in the context of different cell types and during the whole life. For ten years, the nematode Caenorhabditis elegans has emerged as a powerful model to analyze autophagy in physiological or pathological contexts. In this article, we present some of the established approaches and the emerging tools available to monitor and manipulate autophagy in C. elegans, and discuss their advantages and limitations.