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PLoS One. 2011;6(9):e24656. doi: 10.1371/journal.pone.0024656. Epub 2011 Sep 14.

Acute drug treatment in the early C. elegans embryo.

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1
Ludwig Institute for Cancer Research, Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California, United States of America.

Abstract

Genetic and genome-wide RNAi approaches available in C. elegans, combined with tools for visualizing subcellular events with high-resolution, have led to increasing adoption of the early C. elegans embryo as a model for mechanistic and functional genomic analysis of cellular processes. However, a limitation of this system has been the impermeability of the embryo eggshell, which has prevented the routine use of small molecule inhibitors. Here, we present a method to permeabilize and immobilize embryos for acute inhibitor treatment in conjunction with live imaging. To identify a means to permeabilize the eggshell, we used a dye uptake assay to screen a set of 310 candidate genes defined by a combination of bioinformatic criteria. This screen identified 20 genes whose inhibition resulted in >75% eggshell permeability, and 3 that permeabilized embryos with minimal deleterious effects on embryo production and early embryonic development. To mount permeabilized embryos for acute drug addition in conjunction with live imaging, we combined optimized inhibition of one of these genes with the use of a microfabricated chamber that we designed. We demonstrate that these two developments enable the temporally controlled introduction of inhibitors for mechanistic studies. This method should also open new avenues of investigation by allowing profiling and specificity-testing of inhibitors through comparison with genome-wide phenotypic datasets.

PMID:
21935434
PMCID:
PMC3173474
DOI:
10.1371/journal.pone.0024656
[Indexed for MEDLINE]
Free PMC Article
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