A Three-Dimensional In Vivo System for Visualizing Gene Expression

基因如何表达是生物学的一个基本方面。一个未满足的挑战是检查三维基因表达模式在完整的生物体没有中断。目前跟踪体内基因表达的技术要么无法深入渗透，要么无法解决许多基础研究问题。光学相干显微镜（OCM）允许无损的、重复的、定量的、完整生物体内组织和器官的三维可视化。先前的概念验证实验表明，OCM可以使用天然聚合物PHB（聚d -(-)-3-羟基丁酸酯）作为报告基因，对强启动子的体内三维基因表达进行非破坏性成像。这个项目的目标是调查这种方法的敏感性。基因报告基因（PHB）在不同位置和水平表达的转基因植物将被培育出来。然后使用OCM对这些植物进行分析，以可视化报告基因产物，并应用计算机视觉方法以确定该方法的灵敏度。科学，在其最佳状态下，创造未来。科学如何“创造”未来的一个关键部分是通过发现和理解，同时教育他人。该项目的部分支持将用于在梅德福实验室保留少数本科生。此外，还将为2名研究生提供部分支持。为了加强这项工作对当地的影响，还计划在当地一所K-12学校开展外展活动。为了增强这项工作的更广泛的教育影响，这项技术的信息将被纳入课堂教学。为了增强更广泛的科学影响，计划在专业会议上发表报告，并在同行评议的期刊上发表文章。这项工作将运用工程师和计算机视觉专家的知识，为生物学家开发下一代基因分析仪器。因此，这个项目的定位是显著提高科学和技术的认识。

How genes are expressed is a fundamental aspect of biology. An unmet challenge is to examine the three-dimensional gene expression patterns in intact organisms without disruption. Current technologies to follow gene expression in vivo either fail to penetrate deeply enough or provide too poor of a resolution for many basic research questions. Optical coherence microscopy (OCM) allows non-destructive, repetitive, quantitative, in vivo three-dimensional visualization of tissues and organs in intact organisms. Previous proof-of-concept experiments demonstrated that OCM could be used to non-destructively image in vivo, three-dimensional gene expression from a strong promoter using a naturally occurring polymer, PHB (poly-D-(-)-3-hydroxybutyrate) as the reporter gene. The goal of this project is to investigate the sensitivity of this method. Transgenic plants will be generated in which the gene reporter (PHB) is expressed at different levels and at different locations. These plants will then be analyzed using OCM to visualize the reporter gene product, and computer vision methods will be applied with the goal of determining the sensitivity of the method. Science, at its best, creates the future. A key part of how science "creates" the future is through discovery and understanding while educating others. Part of the support for the project will be for retention of a minority undergraduate in the Medford laboratory. In addition, partial support will be provided to two graduate students. To enhance the local impact of the work, an outreach activity to a local K-12 school is also planned. To enhance the wider educational impact of the work, information from the technology will be incorporated into classroom instruction. To enhance the wider scientific impact, presentations at professional meetings and publications in peer-reviewed journals are planned. This work will apply knowledge of engineers and computer vision specialists to develop the next-generation of gene analysis instruments for biologists. As such, this project is firmly positioned to significantly enhance scientific and technological understanding.