P3: RNA INTERFERENCE TO DISSECT STEM CELL POTENTIAL

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Introduction of double stranded (ds) RNA into a cell leads to sequence specific hybridization and degradation of homologous RNA species. This phenomenon, termed RNA interference (RNAi), has emerged as a powerful tool to probe the function of genes in vitro. Compared to antisense mediated gene inhibition, RNAi has the advantage of offering greater sensitivity and specificity, and provides a reliable and reproducible means for gene silencing. RNAi can be achieved in mammalian cells by the cellular introduction of short interfering (si) RNA. PU.1 is a transcription factor that controls the transcription of many critical genes in myeloid cells (granulocytes and monocytes). Silencing of PU.1 expression will be used as proof of principle that by RNAi can successfully block gene expression in hematopoietic cells. This project will develop techniques to apply RNAi to HSCs and to down-regulate key transcription factors in hematopoietic differentiation. Vectors will be developed that express two or more siRNA constructs. Retroviral and adeno associated virus delivery methods will be established for mammalian cells. These approaches will be utilized to silence PU.1 expression in murine myeloid cell lines and in primary bone marrow cells and the consequences on myeloid gene expression, cellular proliferation, and differentiation will be defined in vitro. PU.1 expression will be silenced by RNAi in primary murine HSCs and myeloid cell differentiation, gene expression, and bone marrow repopulation will be assessed in vivo. Silencing of PU.1 expression in HSCs is expected to block myeloid differentiation and gene expression during adult hematopoiesis.