Role of C/EBPepsilon in myeloid differentiation

描述（由申请人提供）： 成熟的中性粒细胞通过一系列定型步骤从造血干细胞产生。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）的出现标志着终末中性粒细胞分化的承诺。C/EBPe在SGP基因的协同上调中起关键作用。小鼠中C/EBPe基因的破坏导致中性粒细胞成熟中的形态和功能缺陷，具有从早幼粒细胞到中幼粒细胞阶段的缺陷性转变。中性粒细胞有双叶核，呼吸爆发活动异常，趋化性和杀菌活性受损。它们缺乏特异性颗粒，并且不能表达编码二级和三级颗粒内容物蛋白的mRNA。小鼠在感染后3-5个月内死亡或死于“骨髓增生”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密切平行。我们已经从C/EBPe -/-小鼠和相应的野生型同窝小鼠的骨髓中制备了2种不同的细胞系，其模拟敲除小鼠中的形态和功能缺陷。使用这些细胞系和原代骨髓细胞，我们建议进一步表征终端中性粒细胞分化的转录调控，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）完成新产生的细胞系的表征并将其建立为C/EBPE -/-表型的可靠模型：2）通过细胞系和原代C/EBPe +/+和-/-骨髓的微阵列分析来鉴定C/EBPeptide的下游靶点;和3）通过逆转录病毒转导用在特定目的2中鉴定的候选基因拯救C/EB肽-/-表型。首先将基因转移到C/EBPe -/-细胞系中，以确定表型逆转。将验证的重要靶点转导到C/EBPe -/-骨髓祖细胞中并移植回C/EBPe -/-小鼠中以评估表型逆转

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