GENES EXPRESSED IN STEM CELLS AND DURING CELL CYCLE

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This proposal has as its central objective, the isolation and characterization of genes that are specifically expressed in primitive reconstituting murine hematopoietic stem cells. Highly enriched populations of such stem cells will be purified by standard positive and negative selection procedures utilizing monoclonal antibodies and fluorescent dyes. Messenger RNA (mRNA) will be isolated from these cells and converted into complementary DNA (cDNA_) that will be used to construct cDNA libraries using modifications of amplification techniques capable of generating representative cDNA libraries from very small numbers of cells. The cDNA libraries will also be normalized to partially equalize the representation of abundant and rare transcripts. Transcripts specific for primitive hematopoietic stem cells will be identified by various display and capture methods to differentiate them from transcripts present in control cell populations such as non hematopoietic cells and more differentiated hematopoietic progenitor cells. Candidate transcripts will be purified and their structure characterized. cDNA libraries will also be prepared from populations of primitive hematopoietic progenitor cells (FDC- P1 cell line as well as primary explanted hematopoietic cells) synchronized at different stages of the cell cycle in hematopoietic stem cells will be isolated and characterized using various cDNA display and capture methods. Finally, the role of specific homeobox gene products ina the regulation of gene expression of primitive hematopoietic progenitor cells will be studied using a rapid high amplitude gene induciton system. By these methods, it is anticipated that a variety of novel gene products can be identified, including cell surface markers, receptors, transcriptions factors and other early response gene products. Such novel gene products could then be used as reagents in various strategies and techniques to obtain more highly enriched, even pure, primitive hematopoietic stem cells as well as committed progenitor cells for cellular biological studies, transplantation and gene transfer/therapy procedures.