斑马鱼为基础的大型在体内小分子屏幕
1Division of Cardiovascular Medicine, Department of Medicine,Vanderbilt University School of Medicine, 2Department of Pharmacology,Vanderbilt University School of Medicine, 3Vanderbilt Institute of Chemical Biology,Vanderbilt University School of Medicine, 4Research Medicine, Veterans Affairs TVHS,Vanderbilt University School of Medicine
Summary
斑马鱼已成为一个强大的<em>在体内</em>表型为基础的药物屏幕和化学遗传分析平台。在这里，我们展示了一个简单，实用的方法大规模使用斑马鱼的胚胎小分子筛选。
Abstract
鉴于他们的小胚胎的大小，快速发展，透明度，繁殖力，以及众多的分子，形态和生理上的相似性，对哺乳动物，已经成为一个强大的斑马鱼<em>在体内</em>表型为基础的药物屏幕和化学遗传分析平台。在这里，我们展示了一个简单，实用的方法大规模使用斑马鱼的胚胎小分子筛选。
Protocol
1）斑马鱼的卵收集化学屏幕一天前的下午，成立10到20的斑马鱼的繁殖坦克。每个水箱装满水从水产养殖系统。使用渔网，将一个成年男性和一到两个成年女性在每个养殖池的内部容器。独立分频器从对方的男性和女性的鱼。标签的笼子，并把他们的盖子。 在屏幕上午上，删除从养殖池的分隔，使斑马鱼进行交配。在未来1小时的课程，让受精卵通过电网秋天在每个内部容器的底部?…
Discussion
规划斑马鱼为基础的化学屏幕时，必须特别注意正在审议的表型，这种表型的背景率的稳健性。诱导表型的化学抑制器的屏幕，这一点尤为重要。例如，对于引起热休克诱导转基因表型，导致表型的条件下再现，必须精确地映射出启动屏幕前，以避免不可接受的高误报率。通过精心规划，斑马鱼化工屏幕，这就需要适度的资本投资，可能会导致的细胞信号转导和生理的新型调制器的发现，以?…
Disclosures
The authors have nothing to disclose.
Materials
- Minimum of 20 pairs of adult zebrafish of desired genotype.
- Fish nets, Breeding tanks, with removable inner container and dividers (Aquatic Habitats).
- Petri dishes (10 cm).
- Plastic tea strainer.
- Wash bottle containg embryo water.
- Disposable polyethylene transfer pipettes.
- Polystyrene 96-well round-bottom assay plates (Corning COSTAR; Lowell, MA).
- Glass Pasteur pipette (Fisher Scientific).
- Manual pipette pump, 10 mL (Bel-Art Products, Pequannock, NJ).
- E3 embryo medium: 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2 , 0.33 mM MgSO4 , containing 0.003% PTU (phenylthio-carbamide, Sigma; St. Louis, MO). PTU can be prepared as a 10x solution by dissolving 0.3-g PTU in 1 L of E3 embryo media. Solutions containing PTU should be protected from light by covering with aluminum foil.
- 12-channel pipettes, 2-20 μL (Eppendorf).
- 12-channel pipettes, 3-300 μL (Eppendorf).
- Disposable polystyrene pipette basin, 50 mL (Fisher Scientific).
- Small molecule library of structurally diverse compounds arrayed in a 96-wll format at 10 mM stock in DMSO. Each master plate is aliquoted into 96-well polypropylene storage plates (Corning), and stored at -80°C until use.
- Aluminum sealing tape for 96-well plates (Nunc, Rochester, NY).
- DMSO (Sigma, St. Louis, MO).
- Basic incubator, 28.5°C (Fisher Scientific).
- Stereomicroscope with transmitted light base (Leica Microsystems, Bannockburn, IL).
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Cite This Article
Hao, J., Williams, C. H., Webb, M. E., Hong, C. C. Large Scale Zebrafish-Based In vivo Small Molecule Screen. J. Vis. Exp. (46), e2243, doi:10.3791/2243 (2010).