Development of a Fluorescence Quantification Assay for Mid to High Throughput Drug Screening

Abstract Background In recent times zebrafish (danio rerio) has been used increasingly for drug screening, target identification, pharmacology and toxicology. Quantifying fluorescence emitted by fluorophores is a method that can be used to measure expression of a gene. I used a novel transgenic zebrafish line to develop a drug screening assay, using fluorescence quantification to assess expression of the atheroprotective klf2a gene, to which the fluorophore is tagged. Materials and Methods The novel transgenic zebrafish line (klf2a:GFP;kdrl:RFP) was developed in our laboratory for my experiments. To establish positive and negative controls for the assay, GFP fluorescence patterns, a surrogate for klf2a expression were observed in two days post fertilization (2dpf) embryos. Images were acquired using a fluorescence stereo-microscope. Green fluorescence was quantified using FIJI (ImageJ) software. GraphPad Prism was used for data analysis. In order to prevent flow-dependent expression of klf2a in developing vasculature, 1 cell stage embryos were injected with 1nl of 0.2mM tnnt2 morpholino into 1 cell stage embryos. No blood flow was established in these embryos. To switch off the flow- dependent expression of klf2a in zebrafish embryonic vasculature blood flow was interrupted at 1 day post fertilization stage, by treating embryos with anaesthetic drug, tricaine. To further examine the non-flow dependent klf2a expression tricaine-only treated embryos were compared with embryos treated with tricaine and lovastatin. Statins like lovastatin are known to upregulate klf2 through non-flow dependent pathways. Feasibility of using a plate-reader was established to further develop the assay for mid to high throughput drug screening. Results I replicated the results to substantiate that klf2a expression was dependent on flow in the vasculature. Lovastatin treatment significantly increased GFP fluorescence in 2dpf embryos as compared to DMSO (dimethyl sulphoxide) treated embryos. I further showed that the assay was reproducible when used for a mid-throughput drug screening.