title: A manual small molecule screen approaching high-throughput using zebrafish embryos.
abstract: Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.
authors.affiliation: Department of Biological Sciences, University of Notre Dame.
authors.forename: Shahram Jevin
authors.initials: SJ
authors.lastname: Poureetezadi
authors.affiliation: Department of Biological Sciences, University of Notre Dame.
authors.forename: Eric K
authors.initials: EK
authors.lastname: Donahue
authors.affiliation: Department of Biological Sciences, University of Notre Dame; rwingert@nd.edu.
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Wingert
mesh_terms: D000818:Animals; D004625:Embryo, Nonmammalian / Q000187:drug effects; D057166:High-Throughput Screening Assays / Q000379:methods*; D017403:In Situ Hybridization / Q000379:methods; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D054852:Small Molecule Libraries / Q000494:pharmacology*; D015027:Zebrafish
chemical_list: D012333:RNA, Messenger; D054852:Small Molecule Libraries