REGULATION OF THE HUMAN CD34 STEM CELL GENE

CD 34在人造血干细胞上特异性表达，而在成熟血细胞上不表达。CD 34+细胞可以重建所有细胞系的正常造血，迄今为止它代表了最明确的人类干细胞标志物;因此，它是干细胞基因表达的模型。以前的研究已经确定了人类和小鼠CD 34的一些启动子元件，但对该基因和其他干细胞基因的调控仍然知之甚少。本项目的目标是继续定义和分析CD 34的控制元件，并表征那些可以指导异源基因干细胞表达的CD 34基因元件。因此，本研究的具体目标是通过以下步骤对CD 34的调控元件进行表征和研究：（1）对人和小鼠CD 34基因进行比较分析，比较DNA序列、DNA酶I超敏位点，并进行交叉杂交研究;（2）鉴定调节干细胞中CD 34基因表达的重要控制元件，在具有人CD 34 PAC克隆的转基因小鼠中测试它们，表征指导干细胞中报告基因表达的CD 34控制元件，并表征调节这些元件的DNA结合蛋白;和（3）使用同源重组将异源基因插入CD 34指导递送到干细胞区室中。这些元素的特性可能导致新的基因治疗载体的开发，以及对干细胞基因调控因子的新理解。

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 best defined human stem cell marker; therefore, it is a model for stem cell gene expression. Previous studies have defined the some of the promoter elements for human and murine CD34, but the regulation of this and other stem cell genes are still very poorly understood. The goal of this project is to continue to define and analyze the control elements of CD34, and to characterize those elements of the CD34 gene which can direct stem cell expression of heterologous genes. Therefore, the specific aims are to characterize and study the regulatory elements of CD34 in the following steps: (1) To perform a comparative analysis of the human and murine CD34 genes, comparing DNA sequence, DNAse I hypersensitive sites, and performing cross hybridization studies; (2) To identify the important control elements regulating CD34 gene expression in stem cells, testing them in transgenic mice with human CD34 PAC clones, characterizing CD34 control elements which direct expression of a reporter gene in stem cells, and characterizing DNA binding proteins which regulate these elements; and (3) To use homologous recombination to insert heterologous genes into CD34 instructs for delivery into the stem cell compartment. 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.