P3: RNA INTERFERENCE TO DISSECT STEM CELL POTENTIAL

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 将双链RNA引入细胞，导致序列特异性杂交和同源RNA物种的降解。这种现象被称为RNA干扰(RNAi)，已经成为在体外探索基因功能的有力工具。与反义介导的基因抑制相比，RNAi具有更高的敏感性和特异性，为基因沉默提供了一种可靠和可重复性的手段。通过细胞内导入短干扰(Si)RNA，可以在哺乳动物细胞中实现RNAi。PU.1是一种转录因子，控制髓系细胞(粒细胞和单核细胞)中许多关键基因的转录。PU.1表达的沉默将被用来证明RNAi可以成功地阻断造血细胞中的基因表达。该项目将开发将RNAi应用于HSCs并下调造血分化中的关键转录因子的技术。将开发表达两个或更多siRNA结构的载体。将建立用于哺乳动物细胞的逆转录病毒和腺相关病毒传递方法。这些 将利用方法沉默PU.1在小鼠髓系细胞系和原代骨髓细胞中的表达，并将在体外确定对髓系基因表达、细胞增殖和分化的影响。PU.1在原代小鼠HSC中的表达将被RNAi沉默，骨髓细胞分化、基因表达和骨髓再生将在体内进行评估。抑制PU1在HSCs中的表达有望阻断成人造血过程中的髓系分化和基因表达。

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.