Effects of genetic mutations and chemical exposures on Caenorhabditis elegans feeding: evaluation of a novel, high-throughput screening assay.
Effects of genetic mutations and chemical exposures on Caenorhabditis elegans feeding: evaluation of a novel, high-throughput screening assay.
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DOI：
10.1371/journal.pone.0001259
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发表时间：
2007-12-05
期刊：
影响因子：
3.7
通讯作者：
Freedman JH
中科院分区：
文献类型：
--
作者：
Boyd WA;McBride SJ;Freedman JH
政府机构已经明确了减少、改进或取代目前以哺乳动物为基础的生物检测方法的必要性，这些方法使用替代物种的检测方法。秀丽隐杆线虫等无脊椎动物因其生命周期短、维持成本低、与高等真核生物高度进化保守而提供了一个有吸引力的选择。线虫咽部是研究神经肌肉功能以及化学物质对神经肌肉活动(即摄食)影响的良好模型。然而，目前的喂养方法是劳动密集型的，而且只是半定量的。在这里，描述了一种高通量分析，它使用流式细胞术来测量线虫在暴露于荧光标记的微球后的大小和肠道荧光，从而测量线虫的摄食。通过定量测定摄食缺陷线虫(EAT突变体)的荧光，以及将野生线虫暴露于神经活性化合物、5-羟色胺和槟榔碱中，验证了该方法的有效性。以前被确定为导致泵送速度缓慢的EAT突变在我们的检测中显示出最低的摄食水平。5-羟色胺暴露后摄食水平的浓度依赖性增加取决于食物的可获得性，而槟榔碱暴露线虫的摄食水平下降，与食物的存在无关。然后利用环境污染物氯化镉和毒死蜱对野生型线虫摄食的影响来演示摄食试验的应用。镉暴露在200微米以上导致摄食水平急剧下降。毒死蜱对线虫的摄食呈浓度依赖性减少，EC50为2微米。线虫荧光微球摄食试验是一种快速、可靠的方法，可用于评估药物、工业化学品或环境制剂的神经毒性效应。这种分析也可能适用于大规模的基因或RNAi筛查，用于识别咽部或其他神经肌肉系统的发育或功能所必需的基因。
Government agencies have defined a need to reduce, refine or replace current mammalian-based bioassays with testing methods that use alternative species. Invertebrate species, such as Caenorhabditis elegans, provide an attractive option because of their short life cycles, inexpensive maintenance, and high degree of evolutionary conservation with higher eukaryotes. The C. elegans pharynx is a favorable model for studying neuromuscular function, and the effects of chemicals on neuromuscular activity, i.e., feeding. Current feeding methodologies, however, are labor intensive and only semi-quantitative. Here a high-throughput assay is described that uses flow cytometry to measure C. elegans feeding by determining the size and intestinal fluorescence of hundreds of nematodes after exposure to fluorescent-labeled microspheres. This assay was validated by quantifying fluorescence in feeding-defective C. elegans (eat mutants), and by exposing wild-type nematodes to the neuroactive compounds, serotonin and arecoline. The eat mutations previously determined to cause slow pumping rates exhibited the lowest feeding levels with our assay. Concentration-dependent increases in feeding levels after serotonin exposures were dependent on food availability, while feeding levels decreased in arecoline-exposed nematodes regardless of the presence of food. The effects of the environmental contaminants, cadmium chloride and chlorpyrifos, on wild-type C. elegans feeding were then used to demonstrate an application of the feeding assay. Cadmium exposures above 200 µM led to a sharp drop in feeding levels. Feeding of chlorpyrifos-exposed nematodes decreased in a concentration-dependent fashion with an EC50 of 2 µM. The C. elegans fluorescence microsphere feeding assay is a rapid, reliable method for the assessment of neurotoxic effects of pharmaceutical drugs, industrial chemicals or environmental agents. This assay may also be applicable to large scale genetic or RNAi screens used to identify genes that are necessary for the development or function of the pharynx or other neuromuscular systems.
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影响因子：
3.4
作者：
Becker, Richard A.;Borgert, Christopher J.;Stott, William T.
通讯作者：
Stott, William T.
DOI：
10.1073/pnas.0400848101
发表时间：
2004-05-25
影响因子：
11.1
作者：
Huang, C;Xiong, CJ;Kornfeld, K
通讯作者：
Kornfeld, K
影响因子：
2.7
作者：
Franks, Dawn M.;Izumikawa, Tomomi;Okkema, Peter G.
通讯作者：
Okkema, Peter G.
影响因子：
56.9
作者：
HORVITZ, HR;CHALFIE, M;EVANS, PD
通讯作者：
EVANS, PD
影响因子：
--
作者：
AVERY, L;HORVITZ, HR
通讯作者：
HORVITZ, HR