Preprint Article Version 1 This version is not peer-reviewed

Live Florescent Staining Platform Drug-Screening and Mechanism-Analysis in Zebrafish for Bone Mineralization

Version 1 : Received: 21 September 2017 / Approved: 21 September 2017 / Online: 21 September 2017 (06:34:11 CEST)

A peer-reviewed article of this Preprint also exists.

Chen, J.-R.; Lai, Y.-H.; Tsai, J.-J.; Hsiao, C.-D. Live Fluorescent Staining Platform for Drug-Screening and Mechanism-Analysis in Zebrafish for Bone Mineralization. Molecules 2017, 22, 2068. Chen, J.-R.; Lai, Y.-H.; Tsai, J.-J.; Hsiao, C.-D. Live Fluorescent Staining Platform for Drug-Screening and Mechanism-Analysis in Zebrafish for Bone Mineralization. Molecules 2017, 22, 2068.

Journal reference: Molecules 2017, 22, 2068
DOI: 10.3390/molecules22122068

Abstract

Currently, drug screening is primarily based on human cell culture for initial high-throughput screening, and subsequently, rodent model to confirm the biological effects. However, the mammalian system is known for time-consuming and highly-cost to be difficult to perform high-throughput drug screening, which exists a critical gap between in vitro cell-based models and the in vivo mammalian models. Therefore, the zebrafish could bridge this gap in preclinical toxicity screening along the drug development pipeline because of its efficiency. We aimed to develop an in vivo zebrafish platform for rapid drug screening. Zebrafish, due to its high genomic conservation with mammals and rapid development and differentiation, it has many advantages, such as short life span, large number of offspring and low cost, easy manipulation for generating transgenic species, to serve as animal model for disease-based research. In 96-well microplates, zebrafish embryos were incubated with small molecular compounds that affected bone mineralization. The level of osteogenic mineralization was evaluated by fluorescent dye staining and quantified by image analysis software. Quantitative real time-PCR (qRT-PCR) was performed to evaluate the biological pathways involved in bone metabolism at the molecular level. The system was validated by demonstrating that response to alendronate and Dorsomorphin in zebrafish. In our study, we screened for 24 compounds within the CYCU-1120~1152 chemical library and identified 3 compounds, pentamidine (CYCU-1140), BML-267 (CYCU-1147), and alendronate (CYCU-1152), increased embryonic mineralization; while 6 compounds, RWJ-60475 (CYCU-1126), levamisole HCL (CYCU-1128), tetramisole HCL (CYCU-1129), fenvalerate (CYCU-1132), NSC-663284 (CYCU-1138), and BML-267ester (CYCU-1148), were inhibitory to bone mineralization. We also found that alendronate enhanced the level of bone mineralization by inhibiting osteoclast-related genes. To sum up, our research showed that zebrafish may have potential to be a drug-screening and mechanism-analysis platform for bone mineralization.

Subject Areas

drug screening; bone mineralization; osteoclast; zebrafish

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