Novel System to Automate Zebrafish Angiogenesis Assays

描述（由申请人提供）： 斑马鱼Danio rerio已被广泛接受为人类疾病的模型。人们对开发斑马鱼作为用于高通量药物发现测定的模式生物有强烈的兴趣。在药物发现中使用斑马鱼的优势包括它们吸收候选药物分子的容易性以及它们与人类的遗传和生理相似性。到目前为止，斑马鱼的高通量筛选仅限于胚胎和非常年幼的幼虫。这是因为研究人员最感兴趣的血管和许多器官最好从动物的侧面观察。一旦鱼鳔膨胀，幼虫通常会直立漂浮，使内部结构难以可视化。在大多数高通量药物发现中使用的多孔微孔板格式中尤其如此。解决这一重要问题的努力集中在使用共聚焦成像技术或强大的图像去卷积软件来组装动物的3-D重建。这些努力没有实现期望的空间分辨率，并且通常需要1至2分钟来对单个井进行成像。物理科学公司(PSI)发明了一种新技术，将适用于已在药物发现实验室使用的标准图像分析系统。PSI的技术将使研究人员能够在微孔阵列中生成老年斑马鱼幼虫的清晰侧视图。通过提供斑马鱼的侧视图，该技术将能够以秒/孔相对于当前技术的分钟/孔生成质量图像。在第I阶段，我们将生成转基因斑马鱼的脉管系统的荧光图像，所述转基因斑马鱼在thefli-1调控序列的控制下表达增强型绿色荧光蛋白（EGFP）。我们将比较具有正常血管发育的幼虫与用VEGF抑制剂处理的幼虫的图像，并且其具有不完全发育的血管。这些实验将作为一个模型，用于在斑马鱼中开发一种全自动、高通量的药物发现试验，以寻找新型血管生成抑制剂。

DESCRIPTION (provided by applicant): The zebrafish, Danio rerio, has become widely accepted as a model for human disease. There is strong interest in developing zebrafish as a model organism for use in high throughput drug discovery assays. Advantages of using zebrafish in drug discovery include the ease with which they absorb candidate drug molecules and their genetic and physiologic similarity to humans. Thus far, high throughput screening in zebrafish has been limited to embryos and very young larvae. This is because the vasculature and many of the organs that are most interesting to researchers are best viewed from the side of the animal. Once the swim bladder inflates the larvae generally float upright making visualization of internal structures difficult. This is especially true in the multiwell microplate formats that are used in most high-throughput drug discovery. Efforts to resolve this important problem have centered on use of confocal imaging techniques or powerful image deconvolution software to assemble 3-D reconstructions of the animal. These efforts have not achieved the desired spatial resolution and typically require 1 to 2 minutes to image a single well. Physical Sciences Inc. (PSI) has invented a novel technology that will be adaptable to standard image analysis systems that are already in use in drug discovery laboratories. PSI's technology will allow researchers to generate clear side-views of older zebrafish larvae in microwell arrays. By providing a side view of the zebrafish, the technology will enable generation of quality images in seconds per well versus minutes per well for current techniques, hi Phase I we will generate fluorescence images of the vasculature of transgenic zebrafish that express Enhanced Green Fluorescent Protein (EGFP) under control of thefli-1 regulatory sequences. We will compare images of larvae with normal vascular development with those of larvae treated with a VEGF inhibitor and which have incompletely developed vasculature. These experiments will serve as a model for Phase II development of a fully-automated, high-throughput, drug-discovery assay in zebrafish for novel angiogenesis inhibitors.