title: High-throughput screening for bioactive molecules using primary cell culture of transgenic zebrafish embryos.
abstract: Transgenic zebrafish embryos expressing tissue-specific green fluorescent protein (GFP) can provide an unlimited supply of primary embryonic cells. Agents that promote the differentiation of these cells may be beneficial for therapeutics. We report a high-throughput approach for screening small molecules that regulate cell differentiation using lineage-specific GFP transgenic zebrafish embryonic cells. After validating several known regulators of the differentiation of endothelial and other cell types, we performed a screen for proangiogenic molecules using undifferentiated primary cells from flk1-GFP transgenic zebrafish embryos. Cells were grown in 384-well plates with 12,128 individual small molecules, and GFP expression was analyzed by means of an automated imaging system, which allowed us to screen thousands of compounds weekly. As a result, 23 molecules were confirmed to enhance angiogenesis, and 11 of them were validated to promote the proliferation of mammalian human umbilical vascular endothelial cells and induce Flk1+ cells from murine embryonic stem cells. We demonstrated the general applicability of this strategy by analyzing additional cell lineages using zebrafish expressing GFP in pancreatic, cardiac, and dopaminergic cells.
authors.affiliation: Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, CA 90095, USA.
authors.forename: Haigen
authors.initials: H
authors.lastname: Huang
authors.forename: Anne
authors.initials: A
authors.lastname: Lindgren
authors.forename: Xinrong
authors.initials: X
authors.lastname: Wu
authors.forename: Ning-Ai
authors.initials: NA
authors.lastname: Liu
authors.forename: Shuo
authors.initials: S
authors.lastname: Lin
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D004625:Embryo, Nonmammalian / Q000166:cytology* / Q000378:metabolism*; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D051379:Mice; D009928:Organ Specificity; D015027:Zebrafish / Q000235:genetics / Q000378:metabolism*
chemical_list: D049452:Green Fluorescent Proteins