REGULATION OF THE HUMAN CD34 STEM CELL GENE

CD34在人的造血干细胞上特异表达，而在成熟血细胞上不表达。CD34+细胞可以重建所有细胞系的正常造血，是迄今为止定义最明确的人类干细胞标记物，是干细胞基因表达的模型。以前的研究已经定义了人和小鼠CD34的一些启动子元件，但对这个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CD34基因的调控元件，并鉴定那些可以指导干细胞表达异源基因的CD34基因元件。因此，本研究的具体目的是通过以下步骤鉴定和研究CD34的调控元件：(1)比较人和小鼠的CD34基因，比较DNA序列和DNase I超敏感部位，并进行交叉杂交研究；(2)识别调控干细胞中CD34基因表达的重要调控元件，在带有人CD34 PAC克隆的转基因小鼠中检测它们，鉴定直接在干细胞中表达报告基因的CD34调控元件，并鉴定调节这些元件的DNA结合蛋白；(3)使用同源重组将异源基因插入CD34指令，将其输送到干细胞室。这些元件的特性可能导致开发新的基因治疗载体，以及对干细胞中调控基因的因素有新的理解。

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.