title: Automated reporter quantification in vivo: high-throughput screening method for reporter-based assays in zebrafish.
abstract: Reporter-based assays underlie many high-throughput screening (HTS) platforms, but most are limited to in vitro applications. Here, we report a simple whole-organism HTS method for quantifying changes in reporter intensity in individual zebrafish over time termed, Automated Reporter Quantification in vivo (ARQiv). ARQiv differs from current "high-content" (e.g., confocal imaging-based) whole-organism screening technologies by providing a purely quantitative data acquisition approach that affords marked improvements in throughput. ARQiv uses a fluorescence microplate reader with specific detection functionalities necessary for robust quantification of reporter signals in vivo. This approach is: 1) Rapid; achieving true HTS capacities (i.e., >50,000 units per day), 2) Reproducible; attaining HTS-compatible assay quality (i.e., Z'-factors of ≥0.5), and 3) Flexible; amenable to nearly any reporter-based assay in zebrafish embryos, larvae, or juveniles. ARQiv is used here to quantify changes in: 1) Cell number; loss and regeneration of two different fluorescently tagged cell types (pancreatic beta cells and rod photoreceptors), 2) Cell signaling; relative activity of a transgenic Notch-signaling reporter, and 3) Cell metabolism; accumulation of reactive oxygen species. In summary, ARQiv is a versatile and readily accessible approach facilitating evaluation of genetic and/or chemical manipulations in living zebrafish that complements current "high-content" whole-organism screening methods by providing a first-tier in vivo HTS drug discovery platform.
authors.affiliation: Department of Cellular Biology and Anatomy, Georgia Health Sciences University, Augusta, Georgia, United States of America.
authors.forename: Steven L
authors.initials: SL
authors.lastname: Walker
authors.forename: Junko
authors.initials: J
authors.lastname: Ariga
authors.forename: Jonathan R
authors.initials: JR
authors.lastname: Mathias
authors.forename: Veena
authors.initials: V
authors.lastname: Coothankandaswamy
authors.forename: Xiayang
authors.initials: X
authors.lastname: Xie
authors.forename: Martin
authors.initials: M
authors.lastname: Distel
authors.forename: Reinhard W
authors.initials: RW
authors.lastname: Köster
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Parsons
authors.forename: Kapil N
authors.initials: KN
authors.lastname: Bhalla
authors.forename: Meera T
authors.initials: MT
authors.lastname: Saxena
authors.forename: Jeff S
authors.initials: JS
authors.lastname: Mumm
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001331:Automation / Q000379:methods; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004625:Embryo, Nonmammalian; D018628:Gene Dosage / Q000502:physiology; D020869:Gene Expression Profiling / Q000379:methods; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter; D057166:High-Throughput Screening Assays / Q000379:methods*; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D009994:Osmolar Concentration; D015203:Reproducibility of Results; D054928:Validation Studies as Topic; D015027:Zebrafish / Q000196:embryology / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria