Role of C/EBPepsilon in myeloid differentiation

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。的 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）标志着终末中性粒细胞的承诺， 分化C/EBPe在SGP基因的协同上调中起关键作用。 小鼠C/EBPe基因的破坏导致中性粒细胞成熟的形态和功能缺陷 从早幼粒细胞到中幼粒细胞阶段的转变有缺陷。中性粒细胞双叶化 细胞核、异常呼吸爆发活动以及趋化性和杀菌活性受损。他们缺乏 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。表型和 C/EBPe -/-小鼠的功能缺陷与继发性粒细胞增多症患者的功能缺陷非常相似， 缺陷我们已经从C/EBPe -/-小鼠的骨髓中制备了2种不同的细胞系， 相应的野生型同窝出生的小鼠，其模拟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转录水平。 终末中性粒细胞分化的调节，特别是C/EBPe在中性粒细胞分化中的作用 成熟计划我们的具体目标是：1）完成新生成细胞的表征 线，并将其建立为C/EBPE -/-表型的忠实模型：2）鉴定下游靶点 C/EBPepsilon的表达通过微阵列分析细胞系和原代C/EBPe +/+和-/-骨髓; 和3）通过用鉴定的候选基因进行逆转录病毒转导来拯救C/EB肽-/-表型 在具体说明目标2时，首先将基因转移到C/EBPe-/-细胞系中，以确定逆转录酶的表达。 表型经验证的重要靶标将被转导到C/EBPe-/-骨髓祖细胞中， 移植回C/EBPe-/-小鼠以评估表型逆转。

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 defectivetransition 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 defectsof 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/EBPepsilonthrough microarray analysis of the cell lines and primary C/EBPe +/+ and -/- bonemarrow; 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 reversalof phenotype. Verified important targets will be transduced into C/EBPe-/- marrow progenitors and transplanted back into C/EBPe-/- mice to assess reversal of phenotype.