PHYSIOLOGICAL ROLE OF ETS GENES IN HEMATOPOIESIS

ETS 基因在造血中的生理作用

• 批准号: 6478158
• 负责人: Makio Ogawa
• 金额: $ 7.67万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6478158
• 关键词: Retroviridae; beta galactosidase; cell differentiation; clone cells; developmental immunology; flow cytometry; gene expression; genetic regulation; genetic transduction; hematopoiesis; hematopoietic stem cells; human tissue; immunocytochemistry; laboratory mouse; phenotype; polymerase chain reaction; transcription factor; transfection /expression vector

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.

最近的研究表明 ETS 家族基因，特别是 ETS1 和 FLI- 1、可能在造血的病理生理学中起重要作用。 然而，该信息主要基于基因敲除分析 小鼠和培养中的人类细胞系的研究。在项目#3中，我们建议 对原代小鼠和人类造血细胞进行直接研究 为了描述 ETS1 和 FLI-1 的生理功能 造血作用。细胞将直接从小鼠或人身上收获，或者 在原代悬浮液或甲基纤维素培养物中制备。在特定目标下 #1 RT-PCR 和免疫组织化学技术将用于研究 代表不同谱系和阶段的细胞的基因表达 造血发育。谱系限制的成熟纯种群 细胞和高度富集的单能和多能细胞群 祖细胞将通过多种技术的组合来制备，包括 荧光激活细胞分选仪 (FACS) 细胞分选和甲基纤维素 克隆细胞培养。最终，我们将通过以下方式分析基因表达： 使用单细胞 RT-PCR 分离单个造血祖细胞。在 具体目标#2 ETS1 的抑制和强制表达的影响 和 FLI-1 基因对造血祖细胞分化的影响 通过使用逆转录病毒转染进行研究。我们将构建向量 含有大肠杆菌β-半乳糖苷酶（β-gal）基因和正义或反义 一个 ETS 家族基因的序列，以便清楚地区分 由非转导细胞转导。富集 FACS 的祖细胞将 在适当的细胞培养下暴露于逆转录病毒载体 条件下，再次分选 β-gal+ 细胞，并将样品置于平板中 在甲基纤维素培养物中进行集落形成。对血统的影响 小鼠细胞的表达与造血功能相关 在各自的基因敲除小鼠中发现了异常。比较 人类细胞的细胞培养研究将提供对 FLI-1和ETS1基因在人类病理生理学中的作用 造血作用。