Role of C/EBPepsilon in myeloid differentiation

C/EBPepsilon 在骨髓分化中的作用

• 批准号: 7018389
• 负责人: Stephanie Halene
• 金额: $ 12.77万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7018389
• 关键词: NOD mouse; SCID mouse; bone marrow; bone marrow transplantation; cell line; developmental genetics; hematopoiesis; microarray technology; myeloid stem cell; neutrophil; transcription factor; transfection

DESCRIPTION (provided by applicant): Mature neutrophils arise from the hematopoietic stem cell via a series of commitment steps. The appearance of the secondary granule proteins (SGP) lactoferrin (LF), transcobalamin I (TCI), neutrophil collagenase (NC) and neutrophil gelatinase (NG) marks the commitment to terminal neutrophil differentiation. C/EBPepsilon (C/EBPe) plays a critical role in the coordinate upregulation of SGP genes. Disruption of the C/EBPe gene in mice leads to morphologic and functional defects in neutrophil maturation with a defective transition from the promyelocyte to the myelocyte stage. The neutrophils have bilobed nuclei, abnormal respiratory burst activity, and impaired chemotaxis and bactericidal activity. They lack specific granules and fail to express mRNAs encoding for secondary and tertiary granule content proteins. The mice die within 3-5 months of infection or from complications of "myeloproliferation". Phenotypic and functional defects of the C/EBPe -/- mice closely parallel those in patients with secondary granule deficiency. We have made 2 different cell lines from the bone marrow of the C/EBPe -/- mice and corresponding wildtype littermates that mimic the morphologic and functional defects in the knockout mice. Using these cell lines and primary marrow cells, we propose to further characterize the transcriptional regulation of terminal neutrophil differentiation and specifically the role of C/EBPe in the neutrophil maturation program. Our specific aims are: 1) To complete the characterization of the newly generated cell lines and establish them as a faithful model of the C/EBPE -/- phenotye: 2) To identify downstream targets of C/EBPepsilon through microarray analysis of the cell lines and primary C/EBPe +/+ and -/- bone marrow; and 3) To rescue the C/EBPepsilon -/- phenotype by retroviral transduction with candidate genes identified in specif aim 2 . Genes will be transferred first into the C/EBPe -/- cell line to determine reversal of phenotype. Verified important targets will be transduced into C/EBPe -/- marrow progenitors and transplanted back into C/EBPe -/- mice to assess reversal of phenotype

描述（由申请人提供）： 成熟的中性粒细胞通过一系列的定型步骤由造血干细胞产生。次级颗粒蛋白（SGP）、乳铁蛋白（LF）、转钴胺素 I（TCI）、中性粒细胞胶原酶（NC）和中性粒细胞明胶酶（NG）的出现标志着中性粒细胞终末分化的承诺。 C/EBPepsilon (C/EBPe) 在 SGP 基因的协调上调中发挥着关键作用。小鼠中 C/EBPe 基因的破坏会导致中性粒细胞成熟的形态和功能缺陷，并导致从早幼粒细胞到粒细胞阶段的过渡有缺陷。中性粒细胞具有双叶细胞核、异常的呼吸爆发活性以及受损的趋化性和杀菌活性。它们缺乏特定的颗粒，并且无法表达编码二级和三级颗粒内容蛋白的 mRNA。小鼠在感染后 3-5 个月内死亡或死于“骨髓增殖”并发症。 C/EBPe -/- 小鼠的表型和功能缺陷与继发性颗粒缺乏症患者的表型和功能缺陷非常相似。我们从 C/EBPe -/- 小鼠和相应野生型同窝小鼠的骨髓中制备了 2 种不同的细胞系，模拟了基因敲除小鼠的形态和功能缺陷。使用这些细胞系和原代骨髓细胞，我们建议进一步表征中性粒细胞终末分化的转录调控，特别是 C/EBPe 在中性粒细胞成熟程序中的作用。我们的具体目标是： 1) 完成新生成的细胞系的表征，并将其建立为 C/EBPE -/- 表型的忠实模型： 2) 通过细胞的微阵列分析来识别 C/EBPepsilon 的下游靶标系和原代 C/EBPe +/+ 和 -/- 骨髓； 3)通过用特定目标2中确定的候选基因进行逆转录病毒转导来拯救C/EBPepsilon -/-表型。基因将首先转移到 C/EBPe -/- 细胞系中以确定表型的逆转。经过验证的重要靶标将被转导到 C/EBPe -/- 骨髓祖细胞中，并移植回 C/EBPe -/- 小鼠中以评估表型的逆转