Recent studies indicate that ETS family genes, particularly ETS1 and FLI- 1, may play significant roles in the pathophysiology of hematopoiesis. This information, however, is based primarily on analysis of gene knockout mice and studies of human cell lines in culture. In project #3 we propose to carry out direct study of primary murine and human hematopoietic cells in order to delineate the physiological functions of ETS1 and FLI-1 in hematopoiesis. The cells will be harvested directly from mice or man, or prepared in primary suspension or methylcellulose culture.
In specific aim #1 RT-PCR and immunohistochemical techniques will be used for study of the gene expression by cells representing different lineages and stages of hematopoietic development. Lineage restricted pure populations of mature cells and highly enriched populations of monopotential and multi-potential progenitors will be prepared by combinations of techniques including Fluorescence-Activated Cell Sorter (FACS) cell sorting and methylcellulose clonal cell culture. Ultimately, we will analyze the gene expression by individual hemopoietic progenitors by use of single cell RT-PCR.
In specific aim #2 the effects of inhibition and forced expression of ETS1 and FLI-1 genes on the differentiation of hematopoietic progenitors will be studied by using retroviral transfection. We will construct vectors contain E-coli beta-galactosidase (beta-gal) gene and sense or antisense sequences of one ETS family gene in order to distinguish clearly the transduced from non-transduced cells. FACS-enriched progenitors will be exposed to the retroviral vectors under appropriate cell culture conditions, sorted again for beta-gal+ cells and the samples will be plate in methylcellulose culture for colony formation. The effects on lineage expression by murine cells will be correlated with the hematopoietic abnormalities seen in their respective gene knockout mice. Comparisons with the cell culture studies of human cells will provide insight into the roles of FLI-1 and ETS1 genes in the pathophysiology of human hematopoiesis.
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