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Methods Cell Biol. 2018;144:185-231. doi: 10.1016/bs.mcb.2018.03.008. Epub 2018 May 1.

Employing the one-cell C. elegans embryo to study cell division processes.

Author information

1
Ludwig Institute for Cancer Research, La Jolla, CA, United States; Department of Cellular & Molecular Medicine, University of California San Diego, La Jolla, CA, United States.
2
Ludwig Institute for Cancer Research, La Jolla, CA, United States; Department of Cellular & Molecular Medicine, University of California San Diego, La Jolla, CA, United States. Electronic address: abdesai@ucsd.edu.

Abstract

The one-cell Caenorhabditis elegans embryo offers many advantages for mechanistic analysis of cell division processes. Conservation of key genes and pathways involved in cell division makes findings in C. elegans broadly relevant. A key technical advantage of this system is the ability to penetrantly deplete essential gene products by RNA interference (RNAi) and replace them with wild-type or mutant versions expressed at endogenous levels from single copy RNAi-resistant transgene insertions. This ability to precisely perturb essential genes is complemented by the inherently highly reproducible nature of the zygotic division that facilitates development of quantitative imaging assays. Here, we detail approaches to generate targeted single copy transgene insertions that are RNAi-resistant, to engineer variants of individual genes employing transgene insertions as well as at the endogenous locus, and to in situ tag genes with fluorophores/purification tags. We also describe imaging assays and common image analysis tools employed to quantitatively monitor phenotypic effects of specific perturbations on meiotic and mitotic chromosome segregation, centrosome assembly/function, and cortical dynamics/cytokinesis.

KEYWORDS:

C. elegans; Cell division; Centrosome; Cytokinesis; Kinetochore; Meiosis; Mitosis

PMID:
29804670
PMCID:
PMC6374127
DOI:
10.1016/bs.mcb.2018.03.008
[Indexed for MEDLINE]
Free PMC Article

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