P3: RNA INTERFERENCE TO DISSECT STEM CELL POTENTIAL

P3：RNA 干扰剖析干细胞潜力

• 批准号: 7610568
• 负责人: Bharat Ramratnam
• 金额: $ 6.03万
• 依托单位: ROGER WILLIAMS HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7610568
• 关键词: Adult; Bone Marrow; Bone Marrow Cells; Cell Differentiation process; Cell Line; Cell Proliferation; Cells; Computer Retrieval of Information on Scientific Projects Database; Dependovirus; Double-Stranded RNA; Funding; Gene Expression; Gene Silencing; Genes; Genetic Transcription; Grant; Hematopoiesis; Hematopoietic; In Vitro; Institution; Mammalian Cell; Mediating; Methods; Mus; Myelogenous; Myeloid Cells; RNA; RNA Interference; Research; Research Personnel; Resources; Sensitivity and Specificity; Small Interfering RNA; Source; Stem cells; Techniques; United States National Institutes of Health; gene function; granulocyte; in vivo; monocyte; tool; transcription factor; vector

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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 将双链（DS）RNA引入细胞，导致序列特定的杂交和同源RNA物种的降解。这种现象称为RNA干扰（RNAi），已成为探测基因在体外功能的强大工具。与反义介导的基因抑制相比，RNAi具有更大的敏感性和特异性的优势，并为基因沉默提供了可靠和可重现的手段。通过简短的干扰（SI）RNA的细胞引入，可以在哺乳动物细胞中实现RNAi。 PU.1是一个转录因子，可控制髓样细胞中许多关键基因的转录（粒细胞和单核细胞）。 PU.1表达的沉默将用作原理证明，RNAi可以成功地阻止造血细胞中的基因表达。该项目将开发技术将RNAi应用于HSC并下调造血分化中的关键转录因子。将开发出表达两个或多个siRNA构建体的向量。将针对哺乳动物细胞建立逆转录病毒和相关病毒递送方法。这些 方法将用于沉默pu.1在鼠髓样细胞系和原代骨髓细胞中的表达以及对髓样基因表达，细胞增殖和分化的后果将在体外定义。 PU.1的表达将被原代鼠HSC中的RNAi沉默，髓样细胞分化，基因表达和骨髓再生将在体内评估。 PU.1在HSC中表达的沉默预计会阻止成年造血期间的髓样分化和基因表达。