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。一个尚未解决的挑战是在不中断的情况下检查完整生物体的三维基因表达模式。目前跟踪体内基因表达的技术要么不够深入，要么对许多基础研究问题提供的解决方案太差。光学相干显微镜(OCM)可以对完整生物体的组织和器官进行无损的、重复的、定量的活体三维可视化。以往的概念验证实验表明，使用天然聚合物PHB(聚-D-(-)-3-羟基丁酸酯)作为报告基因，OCM可以用于体内非破坏性地成像来自强启动子的三维基因表达。本项目的目的是研究这种方法的敏感性。将产生转基因植物，其中基因报告基因(PHB)在不同水平和不同位置表达。然后，将使用OCM对这些植物进行分析，以可视化报告基因产物，并将应用计算机视觉方法，以确定该方法的灵敏度。科学，在最好的情况下，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。对该项目的部分支持将用于在梅德福德实验室保留一名少数族裔本科生。此外，还将向两名研究生提供部分支持。为加强这项工作在当地的影响，还计划向当地一所K-12学校开展外联活动。为了加强这项工作的更广泛的教育影响，来自该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演讲，并在同行评议的期刊上发表文章。这项工作将应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，该项目的定位是显著提高对科学技术的理解。

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.