A High-throughput, High-content, Liquid-based C. elegans Pathosystem
| dc.citation.articleNumber | e58068 | |
| dc.citation.issueNumber | 137 | |
| dc.citation.journalTitle | Journal of Visualized Experiments | |
| dc.contributor.author | Anderson, Quinton L. | |
| dc.contributor.author | Revtovich, Alexey V. | |
| dc.contributor.author | Kirienko, Natalia V. | |
| dc.date.accessioned | 2018-09-27T17:33:27Z | |
| dc.date.available | 2018-09-27T17:33:27Z | |
| dc.date.issued | 2018 | |
| dc.description.abstract | The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to combat multiple drug resistance. This has led to the conclusion that researchers do not only need to find new drugs, but also need to develop new ways of finding them. Amongst the most promising candidate methods are whole-organism, in vivo assays that use high-throughput, phenotypic readouts and hosts that range from Caenorhabditis elegans to Danio rerio. These hosts have several powerful advantages, including dramatic reductions in false positive hits, as compounds that are toxic to the host and/or biounavailable are typically dropped in the initial screen, prior to costly follow up. Here we show how our assay has been used to interrogate host variation in the well-documented C. elegans—Pseudomonas aeruginosa liquid killing pathosystem. We also demonstrate several extensions of this well-worked out technique. For example, we are able to carry out high-throughput genetic screens using RNAi in 24- or 96-well plate formats to query host factors in this host-pathogen interaction. Using this assay, whole genome screens can be completed in only a few months, which can dramatically simplify the task of identifying drug targets, potentially without the need for laborious biochemical purification approaches. We also report here a variation of our method that substitutes the gram-positive bacterium Enterococcus faecalis for the gram-negative pathogen P. aeruginosa. Much as is the case for P. aeruginosa, killing by E. faecalis is time-dependent. Unlike previous C. elegans—E. faecalis assays, our assay for E. faecalis does not require preinfection, improving its safety profile and reducing the chances of contaminating liquid-handling equipment. The assay is highly robust, showing ~95% death rates 96 h post infection. | |
| dc.identifier.citation | Anderson, Quinton L., Revtovich, Alexey V. and Kirienko, Natalia V.. "A High-throughput, High-content, Liquid-based C. elegans Pathosystem." <i>Journal of Visualized Experiments,</i> no. 137 (2018) JoVE: https://doi.org/10.3791/58068. | |
| dc.identifier.digital | jove-protocol-58068 | |
| dc.identifier.doi | https://doi.org/10.3791/58068 | |
| dc.identifier.uri | https://hdl.handle.net/1911/102729 | |
| dc.language.iso | eng | |
| dc.publisher | JoVE | |
| dc.rights | Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. | |
| dc.title | A High-throughput, High-content, Liquid-based C. elegans Pathosystem | |
| dc.type | Journal article | |
| dc.type.dcmi | Text | |
| dc.type.publication | publisher version |
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