[
Methods Cell Biol,
1995]
The genetics of Caenorhabditis elegans provides a convenient experimental entry point into many developmental processes and a powerful tool that can be exploited to characterize interactions among a set of genes regulating a particular pathway. Eventually, though, the study of developmental processes becomes a molecular study of gene regulation. At this level, the determination of the on/off state of a gene requires an understanding of not only its transcriptional state, but also post-transcriptional, translational, and post-translational control mechanisms. Although the vertebrate literature is rich in details of factors that influence these regulatory processes, relatively few of the factors responsible for gene expression in the nematode C. elegans have been characterized. This lag in knowledge reflects both the relatively recent arrival of C. elegans on the list of experimental systems, as well as its general unsuitability for biochemistry. There are no tissue culture cell lines established from C. elegans, and it is difficult to isolate, in large amounts, any homogeneous cell type. Moreover, the impermeable eggshell encasing the embryo and the cuticle encasing the worm make pharmacological studies in intact animals difficult and tedious. Grim as this sounds, progress has been made in C. elegans in the field of gene expression. The sensitivity of techniques has improved and the available molecular tool kit has expanded. The study of individual genes has provided descriptions of several regulatory processes, some general and some gene specific. Our current level of understanding of gene regulation is sufficient to say that C. elegans appears, in general, to be a typical eukaryote. As such, C. elegans is amenable to many of the standard analytical approaches used in other developmental systems. The purpose of this chapter is to review our current state of knowledge of transcription and translation in C. elegans (for a review
[
1994]
Nematodes have been cultured continuously in the laboratory since 1944 when Margaret Briggs Gochnauer isolated and cultured the free-living hermaphroditic species Caenorhabditis briggsae. Work with C. briggsae and other rhabditid nematodes, C. elegans, Rhabditis anomala, and R. pellio, demonstrated the relative ease with which they could be cultured. The culturing techniques described here were developed for C. elegans, but are generally suitable (to varying degrees) for other free-living nematodes. Whereas much of the early work involved axenic culturing, most of these techniques are no longer in common use and are not included here. In the 1970s C. elegans became the predominant research model due to work by Brenner and co-workers on the genetics and development of this species. An adult C. elegans is about 1.5 mm long, and under optimal laboratory conditions has a life cycle of approximately 3 days. There are two sexes, males and self-fertile hermaphrodites, that are readily distinguishable as adults. The animals are transparent throughout the life cycle, permitting observation of cell divisions in living animals using differential interference microscopy. The complete cell lineage and neural circuitry have been determined and a large collection of behavioral and anatomical mutants have been isolated. C. elegans has six developmental stages: egg, four larval stages (L1-L4), and adult. Under starvation conditions or specific manipulations of the culture conditions a developmentally arrested dispersal stage, the dauer larva, can be formed as an alternative third larval stage. Many of the protocols included here and other experimental protocols have been summarized in "The Nematode Caenorhabditis elegans". We also include a previously unpublished method for long-term chemostat cultures of C. elegans. General laboratory culture conditions for nematode parasites of animals have been described, but none of these nematodes can be cultured in the laboratory through more than one life cycle. Marine nematodes and some plant parasites have been cultured xenically or with fungi. Laboratory cultivation of several plant parasites on Arabidopsis thaliana seedlings in agar petri plates has also been reported.