Developing an expression screen for axonal guidance cues

描述（由申请人提供）：发育中的神经系统内的神经元以令人难以置信的精确性和特异性相互连接。神经元过程被环境中的线索引导到正确的目标，这些线索要么吸引它们进入适当的通路，要么阻止它们在不适当的通路上延伸。在过去的十年中，已经确定了几个作为指导线索的信号分子家族。通常情况下，这些线索已被确定使用遗传筛选，通过配对的体外生物测定与生化纯化，或通过选定的候选分子的表征。虽然每种方法都有强大的优势，但它们都不能构成快速，系统，高通量的筛选指导活动。该建议的目标是开发一种实用的轴突排斥剂的体外表达筛选。其目的是在相对较短的时间内使用驱避活性的生物测定来筛选重组蛋白的大型文库。通过阳性选择标准鉴定活性分子，这种方法将补充更传统的正向遗传筛选，后者依赖于功能表型的丧失来检测新的活性。在筛选中发现的候选线索随后可以通过反向遗传和敲除方法测试指导活性。新的轴突引导线索的发现将促进我们对神经元互连的形成和可塑性的机制的理解，并可能最终帮助我们改善受损神经系统中的轴突再生。

DESCRIPTION (provided by applicant): Neurons within the developing nervous system interconnect with incredible precision and specificity. Neuronal processes are guided to their correct targets by cues in the environment that either attract them onto appropriate pathways or discourage them from extending on inappropriate pathways. Several families of signaling molecules that act as guidance cues have been identified over the past decade. Typically, these cues have been identified using genetic screens, through the pairing of in vitro bioassays with biochemical purifications, or by the characterization of selected candidate molecules. Although each approach has powerful advantages, none of them constitute a rapid, systematic, high throughput screen for guidance activities. The goal of this proposal is to develop a practical in vitro expression screen for axonal repellents. The aim would be to screen a large library of recombinant proteins in a relatively short period of time using a bioassay for repellent activity. By identifying active molecules through a positive selection criteria, this approach will complement more traditional forward genetic screens that rely on loss of function phenotypes to detect new activities. Candidate cues discovered in the screen could subsequently be tested for guidance activity by reverse genetic and knockdown approaches. The discovery of novel axonal guidance cues will advance our understanding of the mechanisms underlying the formation and plasticity of neuronal interconnections, and may ultimately help us improve axon regeneration in the damaged nervous system.