Large Scale Zebrafish-Based In vivo Small Molecule Screen

Summary

Zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

Abstract

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to mammals, zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

Protocol

1) Zebrafish Egg Collection On the afternoon prior to the day of the chemical screen, set up 10 to 20 zebrafish breeding tanks. Fill each tank with water from the aquaculture system. Using a fish net, transfer one adult male and one to two adult females to inner container in each breeding tank. Separate the male and female fish from each other with a divider. Label the cages and put a lid over them. On the morning of the screen, remove the dividers from breeding tanks and allow zebrafish to m…

Discussion

When planning a zebrafish-based chemical screen, particular attention must be paid to the robustness of the phenotype under consideration and the background rate of such phenotype. This is particularly important for screens for chemical suppressors of an induced phenotype. For example, for a phenotype caused by heat-shock induction of a transgene, the condition that induces the phenotype reproducibly must be precisely mapped out prior to initiating the screen to avoid unacceptably high false positive rates. With care…

Materials

1. Minimum of 20 pairs of adult zebrafish of desired genotype.
2. Fish nets, Breeding tanks, with removable inner container and dividers (Aquatic Habitats).
3. Petri dishes (10 cm).
4. Plastic tea strainer.
5. Wash bottle containg embryo water.
6. Disposable polyethylene transfer pipettes.
7. Polystyrene 96-well round-bottom assay plates (Corning COSTAR; Lowell, MA).
8. Glass Pasteur pipette (Fisher Scientific).
9. Manual pipette pump, 10 mL (Bel-Art Products, Pequannock, NJ).
10. 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.
11. 12-channel pipettes, 2-20 μL (Eppendorf).
12. 12-channel pipettes, 3-300 μL (Eppendorf).
13. Disposable polystyrene pipette basin, 50 mL (Fisher Scientific).
14. 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.
15. Aluminum sealing tape for 96-well plates (Nunc, Rochester, NY).
16. DMSO (Sigma, St. Louis, MO).
17. Basic incubator, 28.5°C (Fisher Scientific).
18. Stereomicroscope with transmitted light base (Leica Microsystems, Bannockburn, IL).