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Methods. 2014 Aug 1;68(3):529-35. doi: 10.1016/j.ymeth.2014.05.011. Epub 2014 Jun 2.

A high-content assay for identifying small molecules that reprogram C. elegans germ cell fate.

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  • 1Department of Pediatrics, University of Pittsburgh School of Medicine, Children's Hospital of Pittsburgh of UPMC, 4401 Penn Avenue, Pittsburgh, PA 15224, USA.
  • 2Department of Oncology, Department of Medicine, Brody School of Medicine, East Carolina University, Greenville, NC 27834, USA; Lineberger Comprehensive Cancer Center, University of North Carolina-Chapel Hill, Chapel Hill, NC 27599, USA. Electronic address: leemy@ecu.edu.
  • 3Department of Pediatrics, University of Pittsburgh School of Medicine, Children's Hospital of Pittsburgh of UPMC, 4401 Penn Avenue, Pittsburgh, PA 15224, USA. Electronic address: paksc@upmc.edu.

Abstract

Recent breakthrough discoveries have shown that committed cell fates can be reprogrammed by genetic, chemical and environmental manipulations. The germline of the nematode Caenorhabditis elegans provides a tractable system for studying cell fate reprogramming within the context of a whole organism. To explore the possibility of using C. elegans in high-throughput screens (HTS), we developed a high-throughput workflow for testing compounds that modulate cell fate reprogramming. We utilized puf-8; lip-1 mutants that have enhanced MPK-1 (an ERK homolog)/MAP kinase (MAPK) signaling. Wild-type C. elegans hermaphrodites produce both sperm and oocytes, and are thus self-fertile. However, puf-8; lip-1 mutants produce only sperm and are sterile. Notably, compounds that pharmacologically down-regulate MPK-1 (an ERK homolog)/MAP kinase (MAPK) signaling are able to reprogram germ cell fate and restore fertility to these animals. puf-8; lip-1 mutants provide numerous challenges for HTS. First, they are sterile as homozygotes and must be maintained as heterozygotes using a balancer chromosome. Second, homozygous animals for experimentation must be physically separated from the rest of the population. Third, a high quality, high-content assay has not been developed to measure compound effects on germ cell fate reprogramming. Here we describe a semi-automated high-throughput workflow that enables effective sorting of homozygous puf-8; lip-1 mutants into 384-well plates using the COPAS™ BIOSORT. In addition, we have developed an image-based assay for rapidly measuring germ cell reprogramming by measuring the number of viable progeny in wells. The methods presented in this report enable the use of puf-8; lip-1 mutants in HTS campaigns for chemical modulators of germ cell reprogramming within the context of a whole organism.

Copyright © 2014 Elsevier Inc. All rights reserved.

KEYWORDS:

C. elegans; Cell fate; Drug screening; High-content screening; Reprogramming

PMID:
24990146
[PubMed - indexed for MEDLINE]
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