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。 本项目的目标是实施一个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的神经元的方法，并从对表型和功能至关重要的mRNA中特异性地鉴定cDNA。 转基因小鼠表达可降解的绿色荧光蛋白（dGFP）从per1启动子，一个时钟元件基因，或从fos启动子，一个时钟驱动的基因，允许自动分离的视交叉上核神经元表达昼夜节律。 开发使用反义RNA扩增（从单个神经元扩增cDNA的线性机制）和代表性差异分析（差异cDNA减法的高保真方法）对单个表型分选的神经元进行的方法，将允许对它们表达的mRNA进行广泛表征，并对在昼夜节律期间表达变化的mRNA进行特异性鉴定。 第二个目标不是假设mRNA表达的变化必然导致蛋白质的变化，而是减少实践一种新的方法来提高蛋白质印迹的灵敏度，并用它来测试蛋白质表达的节律。 第三个目标是开发一种方法，使用高通量光学方法同时测量来自单个神经元的多种蛋白质，该方法被预测为检测足以分析单个神经元中蛋白质的水平的抗原。

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.