MRNA AND PROTEIN DYNAMICS IN IDENTIFIED NEURONS

在高度异质性的中枢神经系统中，最感兴趣的基因是那些参与每种神经元表型的独特功能，以及与疾病、衰老和损伤相关的功能障碍的基因。这个项目的目标是实施一种可推广的策略来识别这些基因。第一个目标是开发分离表型相似神经元的方法，并从对表型和功能至关重要的mRNAs中特异性地识别cDNA。表达来自时钟元件基因PER1启动子或时钟驱动基因FOS启动子的可降解绿色荧光蛋白(DGFP)的转基因小鼠，可以自动分离表达昼夜节律的视交叉上核神经元。在单个表型分离的神经元上使用反义RNA扩增和代表性差异分析的方法将既可以广泛地表征它们表达的mRNAs，也可以特异性地识别其表达在昼夜节律中变化的mRNAs。第二个目标不是假设mRNA表达的变化必然会导致蛋白质的变化，而是实践一种提高蛋白质印迹灵敏度的新方法，并用它来测试蛋白质表达的节律。第三个目标是开发一种同时测量单个神经元中多个蛋白质的方法，使用高通量光学方法，预计这种方法可以在足以分析单个神经元蛋白质的水平上检测抗原。

In the highly heterogeneous central nervous system, the genes of greatest interest are those involved in the unique functions of each neuronal phenotype, and with their dysfunctions related to disease, aging, and injury. The objective of this project is to implement a generalizable stategy to identify these genes. The first aim is to develop methods to isolate phenotypically similar neurons and specifically identify cDNAs from mRNAs that are critical to phenotype and function. Transgenic mice expressing degradable green fluorescent protein (dGFP) from the per1 promoter, a clock-element gene, or from the fos promoter, a clock-driven gene, allow automated isolation of suprachiasmatic nucleus neurons expressing a circadian rhythm. Developing methods to use antisense RNA amplification, a linear mechanism of cDNA amplification from single neurons, and representational difference analysis, a high fidelity method of differential cDNA subtraction, on single phenotypically sorted neurons will allow both broad characterization of the mRNAs they express and specific identification of mRNAs whose expression changes during the circadian rhythm. Rather than assuming that changes in mRNA expression necessarily lead to protein changes, the second aim will reduce to practice a novel method for improved sensitivity in Western blots and use it to test rhythms in protein expression. The third aim seeks to develop a method to simultaneously measure multiple proteins from single neurons, using a high-throughput optical method that is predicted to detect antigens at levels sufficient for analysis of proteins in single neurons.