Abstract
This protocol describes a procedure for screening small molecules for bioactivity and a genetic approach to target identification using the nematode Caenorhabditis elegans as a model system. Libraries of small molecules are screened in 24-well plates that contain a solid agar substrate. On top of the agar mixture, one small-molecule species is deposited into each well, along with worm food (E. coli), and two third-stage or fourth-stage larval worms using a COPAS (Complex Object Parametric Analyzer and Sorter) Biosort. Three to five days later the plates are screened for phenotype. Images of the wells are acquired and archived using a HiDI 2100 automated imaging system (Elegenics). Up to 2,400 chemicals can be screened per week. To identify the predicted protein target of a bioactive molecule, wild-type worms are mutagenized using ethylmethanesulfonate (EMS). Progeny are screened for individuals resistant to the molecules effects. The candidate mutant target that confers resistance is then identified. Target identification might take months.
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Acknowledgements
We thank Jonathan Hodgkin suggesting the use of CO2 to anesthetise worms. We thank Simon Alfred and Regina Fraser for sharing unpublished results, and the anonymous reviewers for insightful suggestions. P.M., S.R.C. and P.J.R. are Canadian Research Chairs in plant molecular biology, plant genomics and molecular neurobiology, respectively. This work was supported by an NSERC Industrial Grant to P.M., and a CIHR Grant, Premier's Research Excellence Award and awards from the Canadian Foundation for Innovation and Ontario Innovation Trust to P.J.R.
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I hereby declare that I (P.J.R.) have a competing financial interest in the publication of this manuscript in that I own shares and am a scientific advisor in the company (Elegenics Inc.) that made the high-throughput digital imager (HiDI) that is described in the accompanying manuscript. Al Howard and Ed Huston, of Elegenics Inc., also have an obvious competing financial interest in the publication of this manuscript. Karl Johanson is formerly of Elegenics, but is no longer with the company, and Anthony Chan is a sub-contractor for Elegenics but has no competing financial interest. I have included A.H., E.H., K.J., and A.C. as authors on the manuscript because they had substantial intellectual input into the construction of HiDI, which we employ in the described protocol.
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Burns, A., Kwok, T., Howard, A. et al. High-throughput screening of small molecules for bioactivity and target identification in Caenorhabditis elegans. Nat Protoc 1, 1906–1914 (2006). https://doi.org/10.1038/nprot.2006.283
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DOI: https://doi.org/10.1038/nprot.2006.283
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