Lift-off polymer array approach to axon guidance

描述(申请人提供)：在小鼠嗅觉系统中，1000万个嗅觉神经元(OSN)从上皮延伸到嗅球。在球状体内，它们只与1800个肾小球靶点中的一个靶点和突触。这种以肾小球为靶点的OSNs代表了一个有趣的模型系统，可以在其中研究路径发现和目标识别。OSNs用什么分子机制来识别它们在球茎中合适的肾小球？在生物工程研究资助计划的这一应用中，提出了一项跨越神经生物学和物理学的多学科合作努力，将使用新技术来识别OSN使用的指导和目标线索。我们假设，以不同肾小球为靶点的OSN必须表达不同的指导线索，使它们能够区分不同的靶点。为了识别这些线索，我们专注于开发高度敏感的分子工具。首先，我们开发了一种能够扩增少量RNA的技术。第二，我们开发了一种新的微阵列打印方法，大大提高了微阵列技术的灵敏度。高度灵敏的放大技术与微阵列数据采集的显著改进相结合，将为我们提供一个强大的屏幕来识别嗅觉系统中的指导线索。在这项应用中，我们将进一步表征和改进这些基于聚合物的阵列的制造。我们将它们与传统的阵列进行比较，然后使用它们来搜索由OSN表示的轴突指导线索。

DESCRIPTION (provided by applicant): In the mouse olfactory system, 10 million olfactory sensory neurons (OSNs) extend from the epithelium into the olfactory bulb. Within the bulb, they will target and synapse with just one of ~1800 glomerular targets. This targeting of OSNs to glomeruli represents an intriguing model system in which to study pathfinding and target recognition. What are the molecular mechanisms used by OSNs to identify their appropriate glomerulus in the bulb? In this application to the Bioengineering Research Grant program, a multidisciplinary, collaborative effort that spans neurobiology and physics is proposed that will employ new techniques to identify guidance and targeting cues used by OSNs. We hypothesize that OSNs which target different glomeruli must express different guidance cues that enable them to distinguish among various targets. To identify these cues, we have focused on developing highly sensitive molecular tools. First, we have developed a technique capable of amplifying small amounts of RNA. Second, we have developed a novel microarray printing method that greatly improves the sensitivity of the microarray technique. The combination of a highly sensitive amplification technique coupled with a dramatic improvement in microarray data acquisition will provide us with a powerful screen to identify guidance cues in the olfactory system. In this application, we will further characterize and refine the fabrication of these polymer-based arrays. We will compare them against conventional arrays, and then use them to search for axon guidance cues expressed by OSNs.