Abstract
Despite the considerable advantages that C. elegans offers for studying gene function in vivo, this system is quite challenging for in vivo electrophysiological analysis of channel function, particularly in neurons. A major problem is that C. elegans neurons are confined in a pressurized and hard-to-penetrate cuticle. Recently, a method for culturing C. elegans embryonic cells has been developed and numerous researchers have already applied this option to study a variety of native ion channels and transporters using various configurations of the patch-clamp technique. C. elegans embryonic cells are obtained from eggs harvested from synchronized gravid adults and then are dissociated using a combination of enzymatic treatment and manual pipetting. Once plated on a surface covered with peanut lectin, cells adhere and differentiate into neurons, muscle and epithelial cells. Cultured embryonic cells recapitulate the expression of differentiation markers and are found in the culture in proportion to their cell type in the mature embryo. Differentiated cells survive well for at least 2 weeks. It should be noted that postembryonic cells do not appear to be generated in these cultures. Cultures can be used for electrophysiological study, testing of pharmacological sensitivities, and for RNAi. C. elegans cell culture thus constitutes the basis for the application of experimental procedures that are not easily applicable to the intact nematode.
Highlights
The value of C. elegans as a model system stems from the combination of two important facts: 1) basic cellular mechanisms are conserved between C. elegans and higher organisms, and 2) facile application of sophisticated molecular, genetic, and cell biological techniques can be executed against the backdrop of known cellular development, defined anatomy and a sequenced well-annotated genome
In this chapter we review the history of method development, the protocol, and electrophysiological methods for analyses of cultured C. elegans cells
The same results can be reproduced in vitro (Figure 2D; Bianchi et al, 2004). These results indicate that mec-4(+ and d)-expressing neurons in culture differentiate and behave to mec-4-expressing touch neurons in vivo (Bianchi et al, 2004)
Summary
The value of C. elegans as a model system stems from the combination of two important facts: 1) basic cellular mechanisms are conserved between C. elegans and higher organisms, and 2) facile application of sophisticated molecular, genetic, and cell biological techniques can be executed against the backdrop of known cellular development, defined anatomy and a sequenced well-annotated genome. Manipulation of the extracellular environment and the administration of drugs through cuticle and gut barriers have remained challenges in live nematode analyses Several of these experimental limitations can be overcome using C. elegans cell culture. Leung and colleagues reported that when the intestinal precursor E blastomeres were cultured in vitro they could differentiate into epithelial cells that produced apical adherens junctions, expressed proteins in a polarized manner and surrounded an extracellular space to generate a structure resembling an intestinal lumen (Leung et al, 1999). Buechner and colleagues described cells morphologically resembling canal cells following dissociation and culture of embryonic cells (Buechner et al, 1999) They observed other cell types appearing to differentiate into neurons, muscles and epithelial cells. This work led to the cell culture protocol described by Christiansen and colleagues that is similar to that outlined below (Christensen et al, 2002)
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