Imaging in Developmental Toxicology

描述(申请人提供)：由BioInVision，Inc.开发的冷冻成像和荧光报告基因转基因小鼠将被用于确定由于中毒暴露而导致的甚至轻微或罕见的发育异常。通过交替切片和成像，冷冻成像系统将获得3D、高分辨率的解剖彩色和细胞荧光图像。低温成像系统由一个具有特殊成像特性的大工作台电动恒温器、一个改装的明场/荧光显微镜和一个机器人XYZ成像系统定位器组成，所有这些都是由控制系统完全自动化的。为了实现高吞吐量，我们将修改系统，一次从多达8-50个胚胎的阵列中获取图像。现有的大量荧光报告基因转基因小鼠突出各种组织(平滑肌细胞、内皮细胞、神经元等)。以及新出现的转基因模型，它们荧光地报告了对毒性压力的基因反应上调。有了冷冻成像，我们将获得微米级的分辨率、颜色和荧光体积，这些将显示这些工程小鼠在中毒暴露后的微妙变化。我们将开发3D图像可视化/分析工具，使我们能够探索虚拟胚胎的发育过程中的细微变化。在第一阶段，我们将论证使用在平滑肌肉中大量表达EGFP的胚胎来描绘胎儿血管、呼吸和胃肠道系统的可行性。在孕妇暴露于TCDD后，我们将通过传统的手动解剖和冷冻成像来分析胎儿，并确定冷冻成像检测粗略和细微变化的能力。由于数据是数字化的，因此将有机会对胚胎进行计算比较。如果这一常规方法成功，我们将使用荧光报告小鼠电池和冷冻成像进行毒理学研究，提供更高的敏感性和机制信息。与公共健康相关：我们将利用一种新的成像技术和转基因小鼠来创建一种非常敏感的方法来评估毒素对发育中的胚胎的影响。这项研究将帮助制药公司和环境科学家筛选微妙的、罕见的毒性影响，并确定一般的作用机制。

DESCRIPTION (provided by applicant): Cryo-imaging, as developed by BioInVision, Inc., and fluorescent reporter gene transgenic mice will be used to determine even subtle or rare developmental abnormalities resulting from toxic exposures. By alternately sec- tioning and imaging, the cryo-imaging system will acquire 3D, high-resolution anatomical color and cellular fluorescence images. The cryo-imaging system consists of a large stage, motorized cryostat with special fea- tures for imaging; a modified, bright-field/fluorescence microscope; and a robotic xyz imaging system posi- tioner, all of which are fully automated by a control system. To achieve high throughput, we will modify the sys- tem to acquire images from arrays of as many as 8-50 embryos at a time. There are a very large number of existing fluorescent-reporter-gene transgenic mice which highlight various tissues (smooth muscle cells, endo- thelial cells, neurons, etc.) and emerging transgenic models which fluorescently report upregulation of gene response to toxic stress. With cryo-imaging, we will obtain micron-scale resolution, color and fluorescent vol- umes which will show subtle changes in these engineered mice as a result of toxic exposure. We will develop 3D image visualization/analysis tools allowing us to probe virtual embryos for subtle changes in development. In Phase I, we will demonstrate feasibility using embryos which abundantly expresses EGFP in smooth muscle delineating the fetal vascular, respiratory, and GI systems. Following maternal exposure to TCDD, which is known to affect vascular development, we will analyze fetuses both by conventional manual dissection and by cryo-imaging, and determine the ability of cryo-imaging to detect both gross and subtle variations. Because data is digital, there will be opportunities for computational comparisons of embryos. If this general approach is successful, we will use batteries of fluorescent reporter mice and cryo-imaging to perform toxicology studies providing improved sensitivity and mechanistic information. PUBLIC HEALTH RELEVANCE: We will utilize a new imaging technology and genetically engineered mice to create a very sensitive method for assessing effects of toxins on developing embryos. This research will help drug companies and environmental scientists screen for subtle, rare toxicity effects and to determine general mechanisms of action.