Burns Andrew R, Kwok Trevor C Y, Howard Al, Houston Ed, Johanson Karl, Chan Anthony, Cutler Sean R, McCourt Peter, Roy Peter J
Department of Medical Genetics and Microbiology, The Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, M5S 1A8, Canada.
Nat Protoc. 2006;1(4):1906-14. doi: 10.1038/nprot.2006.283.
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.
本方案描述了一种筛选具有生物活性的小分子的程序以及一种利用线虫秀丽隐杆线虫作为模型系统进行靶点鉴定的遗传学方法。小分子文库在含有固体琼脂底物的24孔板中进行筛选。在琼脂混合物之上，使用COPAS（复杂物体参数分析仪和分选仪）生物分选仪将一种小分子物质与蠕虫食物（大肠杆菌）以及两条第三阶段或第四阶段的幼虫放入每个孔中。三到五天后，对平板进行表型筛选。使用HiDI 2100自动成像系统（Elegenics）获取并存档孔的图像。每周最多可筛选2400种化学物质。为了鉴定生物活性分子的预测蛋白质靶点，使用甲基磺酸乙酯（EMS）对野生型蠕虫进行诱变。筛选后代中对该分子效应具有抗性的个体。然后鉴定赋予抗性的候选突变靶点。靶点鉴定可能需要数月时间。
