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.
