REGULATION OF THE HUMAN CD34 STEM CELL GENE

CD 34在人造血干细胞上特异性表达， 成熟的血细胞。 CD 34+细胞可重建正常造血 到目前为止，它代表了唯一一个明确的人类 干细胞标记物;因此，它是干细胞基因表达模型。 以前的研究已经确定了CD 34的启动子，以及鉴定了 CD 34基因3'端的一个大片段，含有一个增强子， 指导GD 34在细胞系中的特异性表达。 这个目标 项目是进一步定义和分析CD 34的控制要素， 为了表征CD 34启动子和增强子的元件， 异源基因的直接干细胞表达。 表征 这些元件的组合可能会导致新的基因载体的开发， 治疗，以及新的理解的因素，调节基因， 干细胞 因此，具体的目的是表征和研究监管 在以下步骤中分离和表征CD 34的元件： 包含功能和组织的精确基因组序列 ⑶ 34 3'增强子的特异性元件;（2）为了证明⑶ 34 3'增强子的特异性元件是CD 34 3'增强子的特异性元件， 在细胞系中控制CD 34基因表达的相同元件调节 它在正常造血细胞中的表达，表明这些 元件调节转基因小鼠和正常人中CD 34的表达 （3）分离最小的CD 34启动子/增强子，其将 在干细胞中直接表达报告基因;和（4） 表征与CD 34 3'相互作用的转录因子 目的（1）和（2）中定义的增强子元件，以控制 CD34。 这些研究应有助于更好地了解 干细胞基因的调控，以及鉴定 调节它们的转录因子。 此外，这些实验 还应确定指导茎干生长所需的最低限度的调控因素， 细胞表达外源基因，这将导致新的应用， 基因治疗

CD34 is expressed specifically on human hematopoietic stem cells and not on mature blood cells. CD34+ cells can reconstitute normal hematopoiesis of all cell lines, and to date it represents the only well defined human stem cell marker; therefore, it is a model for stem cell gene expression. Previous studies have defined the promoter for CD34, as well as identified a large fragment 3' of the CD34 gene containing an enhancer which appears to direct specific expression of GD34 in cell lines. The goal of this project is to further define and analyze the control elements of CD34, and to characterize those elements of the CD34 promoter and enhancer which can direct stem cell expression of heterologous genes. The characterization of these elements could lead to the development of new vectors for gene therapy, as well as new understanding of the factors regulating genes in stem cells. Therefore, the specific aims are to characterize and study the regulatory elements of CD34 in the following steps: (l) To isolate and characterize the precise genomic sequences comprising the functional and tissue specific elements of the CD34 3' enhancer; (2) To demonstrate that the same elements which control CD34 gene expression in cell lines regulate its expression in normal hematopoietic cells, demonstrating that these elements regulate CD34 expression in transgenic mice and in normal human CD34+ cells; (3) To isolate the minimal CD34 promoter/enhancer which will direct expression of a reporter gene in stem cells; and (4) To characterize the transcription factor(s) which interact with the CD34 3' enhancer elements defined in aims (l) and (2) to control the expression of CD34. These studies should lead to a better understanding of the regulation of stem cell genes, as well as the identification of the transcription factors which regulate them. In addition, these experiments shall also define the minimal regulatory elements necessary to direct stem cell expression of foreign genes, which should lead to new applications in gene therapy.