Flow Cytometry: Digital Processing of Molecular Information

本项目涉及设计、实施和评估用于真核细胞细胞酶学和组织化学分析的流式细胞仪仪器和操作程序。它分为两个子项目。第一个涉及第二代数字数据采集和脉冲处理系统的开发，用于基于脉冲形状特征的细胞实时分析和分类。第二个子项目涉及开发新的基因表达系统，用于转基因真核细胞的分子和细胞分析。然后，我们打算利用这些技术根据转基因表达水平对转化的植物细胞核进行流动分类选择，最终用于构建代表微观小组织发育调节基因的cDNA文库。本研究的意义如下。在第一种情况下，我们之前的工作已经表明，真核细胞的重要信息包含在这些细胞在流式细胞术分析时产生的波形形状中。目前的流式细胞仪无法提取这种基于形状的信息。所提出的第二代机器基于脉冲形状信息分析和分类细胞的能力将提供关于细胞子结构的重要知识。这将有助于提高对活细胞基本细胞生物学的理解。在第二种情况下，当这些细胞散布在其他细胞类型中时，科学家分析不同细胞类型中基因表达的能力就会受到阻碍。在这个项目中提出的分子方法应该为从任何期望的细胞类型中分离转录本提供一般的手段。这将极大地促进发育和细胞类型特异性基因表达研究的进展。

This project involves the design, implementation, and evaluation of flow cytometric instrumentation and operating procedures for use in the cytoenzymological and histochemical analysis of eukaryotic cells. It is divided into two subprojects. The first involves the development of a second-generation digital data acquisition and pulse processing system for the real-time analysis and sorting of cells based on pulse shape features. The second subproject involves the development of novel gene expression systems for use in the molecular and cellular analysis in flow of transgenic eukaryotic cells. We then intend to employ these techniques for the selection through flow sorting of transformed plant nuclei according to the level of transgene expression, ultimately directed at the construction of cDNA libraries representing the developmentally-regulated genes of tissues that are microscopically-small. The significance of this work is as follows. In the first case, our previous work has shown that important information about eukaryotic cells is contained within the waveform shapes produced by these cells produced when they are subjected to analysis using flow cytometry. Present flow cytometric instrumentation cannot extract this shape-based information. The capability of the proposed second generation machine to analyze and sort cells based on pulse shape information will provide important knowledge about cellular substructure. This will lead to improved understanding of the basic cellular biology of living cells. In the second case, the ability of scientists to analyze the expression of genes within different cell types is hampered when these cells are interspersed within other cell types. The molecular methods proposed in this project should provide general means for isolation of transcripts from any desired cell type. This should greatly facilitate advances in the study of developmental- and cell type-specific gene expression.