COBRE: RW HOSP: P3: RNA INTERFERENCE TO DISSECT STEM CELL POTENTIAL

COBRE：RW HOSP：P3：RNA 干扰剖析干细胞潜力

• 批准号: 7382034
• 负责人: Bharat Ramratnam
• 金额: $ 17.9万
• 依托单位: ROGER WILLIAMS HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7382034
• 关键词: COBRE; RW; HOSP; P3; RNA

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. Recently, RNAi has been applied to several models of malignant disease and holds the promise of becoming a therapeutic modality in oncology. All mature cellular elements of blood are derived from hematopoietic stem cells (HSCs) and gene transcription is a key regulatory mechanism in hematopoietic differentiation. PU.1 is an its transcription factor that controls the transcription of many critical genes in myeloid cells (granulocytes and monocytes). Genetic disruption of PU.1 in mice abrogated fetal myelopoiesis, but because PU.1 disruption caused perinatal lethality, its role could not be defined in adult hematopoiesis. Silencing of PU.1 expression will be used as proof of principle that by RNAi can successfully block gene expression in hematopoietic cells. This proposal 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. These approaches of silencing PU.1 expression in murine cell lines and primary HSCs will provide methods to manipulate gene expression in normal hematopoiesis and may powerful new therapeutic tools for leukemia.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。将双链（ds）RNA引入细胞导致同源RNA种类的序列特异性杂交和降解。这种现象，称为RNA干扰（RNAi），已成为一个强大的工具，探测基因的功能在体外。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，并提供了可靠和可重复的基因沉默手段。RNAi可以通过短干扰（si）RNA的细胞引入在哺乳动物细胞中实现。最近，RNAi已被应用于几种恶性疾病模型，并有望成为肿瘤学的治疗方式。血液中所有成熟的细胞成分都来源于造血干细胞（HSC），基因转录是造血分化的关键调控机制。PU.1是其转录因子，其控制骨髓细胞（粒细胞和单核细胞）中许多关键基因的转录。 小鼠中PU.1的遗传破坏废除了胎儿骨髓生成，但由于PU.1破坏导致围产期致死，因此无法确定其在成人造血中的作用。PU.1表达的沉默将被用作通过RNAi可以成功阻断造血细胞中基因表达的原理的证明。该提案将开发将RNAi应用于HSC并下调造血分化中的关键转录因子的技术。将开发表达两种或更多种siRNA构建体的载体。将为哺乳动物细胞建立逆转录病毒和腺相关病毒递送方法。这些方法将用于沉默鼠骨髓细胞系和原代骨髓细胞中的PU.1表达，并将在体外确定对骨髓基因表达、细胞增殖和分化的影响。PU.1表达将通过RNAi在原代鼠HSC中沉默，并且骨髓细胞分化、基因表达和骨髓再增殖将在体内评估。预期HSC中PU.1表达的沉默会阻断成人造血过程中的髓样分化和基因表达。这些在鼠细胞系和原代HSC中沉默PU.1表达的方法将提供在正常造血中操纵基因表达的方法，并且可能是白血病的强有力的新治疗工具。