REGULATION OF THE HUMAN CD34 STEM CELL GENE

CD34在人造血干细胞上特异性表达，而不在成熟血细胞上表达。CD34+细胞可以重建所有细胞系的正常造血功能，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。先前的研究已经确定了人类和小鼠CD34的一些启动子元件，但是对这个和其他干细胞基因的调控仍然知之甚少。该项目的目标是继续定义和分析CD34的控制元件，并表征CD34基因中可以指导干细胞异种基因表达的元件。因此，具体目的是通过以下步骤对CD34的调控元件进行表征和研究：(1)对人、鼠CD34基因进行比较分析，比较DNA序列、DNAse I超敏位点，并进行交叉杂交研究；(2)鉴定干细胞中调节CD34基因表达的重要控制元件，在人CD34 PAC克隆转基因小鼠中进行检测，表征在干细胞中直接表达报告基因的CD34控制元件，并表征调控这些元件的DNA结合蛋白；(3)利用同源重组将外源基因插入CD34指令中，将其传递到干细胞区室。这些元素的表征可能导致基因治疗的新载体的发展，以及对干细胞中调节基因的因素的新认识。

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.