In Situ Hybridization Assay‐Based Small‐Molecule Screening in Zebrafish

Lili Jing1, Ellen M. Durand1, Catherine Ezzio2, Stephanie M. Pagliuca3, Leonard I. Zon4

1 These authors contributed equally to this work., 2 Muhlenberg College, Allentown, Pennsylvania, 3 Department of Evolutionary Anthropology, Duke University, Durham, North Carolina, 4 Howard Hughes Medical Institute, Chevy Chase, Maryland
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch110236
Online Posting Date:  June, 2012
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Abstract

In vitro biochemical and cell‐based small‐molecule screens have been widely used to identify compounds that target specific signaling pathways, but the identified compounds frequently fail at the animal testing stage, largely due to the in vivo absorption, metabolism, and toxicity properties of the chemicals. Zebrafish has recently emerged as a vertebrate whole‐organism model for small‐molecule screening. The in vivo bioactivity and specificity of compounds are examined from the very beginning of zebrafish screens. In addition, zebrafish is suitable for large‐scale chemical screens, similar to many cellular assays. This protocol describes an approach for in situ hybridization (ISH)‐based chemical screening in zebrafish, which, in principle, can be used to screen any gene product. The described protocol has been used to identify small molecules affecting specific molecular pathways and biological processes. It can also be adapted to zebrafish screens with different readouts. Curr. Protoc. Chem. Biol. 4:143‐160 © 2012 by John Wiley & Sons, Inc.

Keywords: zebrafish; in situ hybridization; small molecule screen; drug discovery; in vivo

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Zebrafish Embryo Collection
  • Basic Protocol 2: Zebrafish Embryo Preparation and Chemical Treatment
  • Basic Protocol 3: In Situ Hybridization Staining of Chemical‐Treated Embryos
  • Support Protocol 1: Synthesis of an Antisense RNA Probe
  • Alternate Protocol 1: Chemical Treatment of Embryos in 96‐Well Mesh Filter Plates
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Zebrafish Embryo Collection

  Materials
  • Zebrafish (a wild‐type strain or a specific mutant strain, depending on the purpose of the screen)
  • E3 buffer (see recipe)
  • Breeding vessel (iSPAWN, Tecniplast)
  • Sieve
  • 100‐ or 150‐mm petri dishes

Basic Protocol 2: Zebrafish Embryo Preparation and Chemical Treatment

  Materials
  • Embryos (collected from protocol 1) plated several hours before the desired stage, considering the timing of transferring and plating embryos
  • E3 buffer (see recipe)
  • Chemical library (e.g., Chembridge DIVERSet)
  • Dimethyl sulfoxide (DMSO)
  • Pronase (Roche, cat. No. 11‐459‐643‐001; dissolve in E3 buffer to 2.5 mg/ml)
  • PBT (see recipe)
  • 4% PFA (see recipe)
  • Methanol
  • Microscope (e.g., Zeiss Stereo Discovery.V8)
  • Transfer pipets (Fisher Scientific, cat. no. 13‐711‐5AM)
  • Multi‐well plates (48‐well flat‐bottom plates, Falcon, cat. no. 353078, or 96‐well flat‐bottom plates, Costar, cat. no. 3596)
  • Multi‐channel vacuum wand (optional, e.g., from V& P Scientific)
  • Multichannel pipets
  • Liquid handling robot, optional
  • Parafilm

Basic Protocol 3: In Situ Hybridization Staining of Chemical‐Treated Embryos

  Materials
  • Embryos, fixed and dehydrated ( protocol 2)
  • Methanol
  • PBT (see recipe)
  • Proteinase K solution (Roche, cat. no. 03115828001; dilute in PBT to 10 µg/ml before use)
  • 4% PFA (see recipe)
  • 25% glutaraldehyde (Sigma, cat. no. G4004)
  • Hybe+ (see recipe)
  • DIG‐labeled RNA probe ( protocol 4)
  • Hybe (see recipe)
  • 2× SSC (see recipe)
  • 0.2× SSC (see recipe)
  • Blocking solution (see recipe)
  • Anti‐DIG‐AP antibody (Roche, cat. no. 11 093 274 910)
  • Prestain solution (see recipe)
  • Staining buffer (see recipe)
  • Stop solution (see recipe)
  • 96‐well mesh filter plate and 96‐well receiver plate (Millipore, cat. no. MANM10010)
  • 1.5‐cup air‐tight container (Lock n' Lock, 4.3 × 5.9 × 1.8–in.)
  • Transfer pipets (Fisher Scientific, cat. no. 13‐711‐5AM)
  • Belly dance rotator (STOVALL Life Science)
  • 70°C incubator (Thermo Electron)
  • Aluminum foil
  • Spot plates in white porcelain, 12 cavities (Fisher, cat. no. S337241)
  • Parafilm
  • Microscope (Zeiss Stereo Discovery.V8)
  • 96‐well round‐bottomed plate (BD, cat. no. 353227)

Support Protocol 1: Synthesis of an Antisense RNA Probe

  Materials
  • Vector with the cDNA template for the probe (vector should contain a T3, T7, or SP6 RNA polymerase promoter around the cDNA insert)
  • Restriction enzyme digestion reagents (New England Biolabs)
  • Agarose
  • 1× TBE (see recipe)
  • Phenol:chloroform:isoamyl alcohol (25:24:1; Sigma, cat. no. P2069)
  • Chloroform:isoamyl alcohol (24:1; Sigma, cat. no. C0549)
  • 3 M sodium acetate pH 5.5 (Ambion, cat. no. 9740)
  • Ethanol
  • 70% (v/v) ethanol in DEPC H 2O
  • DEPC H 2O (see recipe)
  • DIG RNA labeling mixture including:
    • 10× transcription buffer (Roche, cat. no. 14735400)
    • NTP DIG labeling mix labeling mixture (Roche, cat. no. 11277073910)
    • Appropriate RNA polymerase (T3, Promega, cat. no. P207C; T7, Promega, cat. no. P207B; SP6 Promega, cat. no. P207A)
    • RNAse inhibitor (RNasein, Promega, cat. no. N2511, 40 U/µl)
    • RNase‐free DNase I (Roche, cat. no. 04716728001, 10 U/µl)
  • 1 M lithium chloride
  • 1.5‐ml microcentrifuge tubes
  • Electrophoresis apparatus
  • Centrifuge
  • Vortex mixer
  • −20°C freezer
  • NanoDrop spectrophotometer (Thermo Scientific, NanoDrop 2000)
  • 37°C incubator
NOTE: To avoid contamination, be sure to use clean tips and make transfers into clean tubes. Unless they are incubating, keep tubes on ice as much as possible.

Alternate Protocol 1: Chemical Treatment of Embryos in 96‐Well Mesh Filter Plates

  • 96‐well mesh filter plate (Pion, cat. no. PN 120655)
  • 96‐well receiving plate (Millipore, cat. no. MATRNPS50)
  • Multiscreen single‐well culture tray (Millipore, cat. no. MAMCS0110)
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Figures

Videos

Literature Cited

Literature Cited
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   Patton, E.E. and Zon, L.I. 2001. The art and design of genetic screens: zebrafish. Nat. Rev. Genet. 2:956‐966.
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   White, R.M., Cech, J., Ratanasirintrawoot, S., Lin, C.Y., Rahl, P.B., Burke, C.J., Langdon, E., Tomlinson, M.L., Mosher, J., Kaufman, C., Chen, F., Long, H.K., Kramer, M., Datta, S., Neuberg, D., Granter, S., Young, R.A., Morrison, S., Wheeler, G.N., and Zon, L.I. 2011. DHODH modulates transcriptional elongation in the neural crest and melanoma. Nature 471:518‐522.
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   Yu, P.B., Hong, C.C., Sachidanandan, C., Babitt, J.L., Deng, D.Y., Hoyng, S.A., Lin, H.Y., Bloch, K.D., and Peterson, R.T. 2008. Dorsomorphin inhibits BMP signals required for embryogenesis and iron metabolism. Nat. Chem. Biol. 4:33‐41.
   Zon, L.I. and Peterson, R.T. 2005. In vivo drug discovery in the zebrafish. Nat. Rev. Drug Discov. 4:35‐44.
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