Analyses of a hematopoietic regulatory region of transcription factor GATA-2 gene and an application of the region to in vitro proliferation of hematopoietic stem cells.

1.造血干细胞增殖腺病毒载体的构建。为了生成感染性病毒载体，我采用了基于Cre/ loxp介导重组的方法（AdMax^<TM>, Microbix Biosystems Inc.）。构建腺病毒载体的一种常用方法是依赖于两个独立载体之间的体内同源重组，其中一个是修饰的病毒基因组载体，另一个是用于引入感兴趣基因的穿梭载体。然而，在这种情况下，同源重组效率很低，而cre介导的loxP重组效率极高。作为实验的第一步，将mGATA-2 cDNA克隆到穿梭载体2中。转录因子GATA-2基因的造血调控区分析已经确定了GATA-2造血表达必需的5 kbp基因组区域，该区域位于转录起始位点145 - 150 kbp之间。在深入研究了包含GATA-2位点的酵母人工染色体（YAC）的一系列缺失后，我能够定位这个离散的…更多的元素。虽然YAC能够分析远程基因组相互作用，但操纵YAC固有的困难使它们不适合对小区域感兴趣的详细分析。因此，我设计了-150必需区的DNA片段共稳定转染系统，使该片段的多个拷贝链接到指导GFP表达的启动子上。在该系统中，我们可以很容易地通过流式细胞分析检测基本区域的活性，而不需要任何细胞染色。该区域的一系列内部缺失显示，NcoI-SpeI的核心活动为1.5 kbp，位于基本区域的中间（附录，图4）。1.5 kbp片段的核苷酸序列揭示了几个完美的调节因子结合候选位点，包括SCL和AML-1。我预计，对核心元件的进一步解剖可能会导致鉴定出一种新的中胚层因子，然后我们将对其进行更详细的研究。一旦进一步分析证实各反式作用因子对GATA-2调控的意义，这些因子也可应用于造血干细胞体外扩增系统。少

1.Construction of an adenovirus vector aiming for the proliferation of hematopoietic stem cells.To generate infectious viral vector, I employed a method based on Cre/loxP-mediated recombination (AdMax^<TM>, Microbix Biosystems Inc.). A common method for constructing adenovirus vectors relies on in vivo homologous recombination between two separate vectors, one of which is a modified viral genomic vector, and the other of which is a shuttle vector used to introduce the gene of interest. However, the homologous recombination efficiency in this case is very low, while Cre-mediated loxP recombination is extremely efficient. As the first experimental step, mGATA-2 cDNA was cloned into the shuttle vector.2.Analyses of a hematopoietic regulatory region of transcription factor GATA-2 geneAn essential 5 kbp genomic region for GATA-2 hematopoietic expression, which is located between 145 and 150 kbp from the transcription initiation site, has been identified. I was able to localize this discrete … More element after intensively examining a series of deletions of a yeast artificial chromosome (YAC) harboring the GATA-2 locus. While the YAC enables analyses of long-range genomic interactions, the difficulties inherent in manipulating YACs makes them inappropriate for detailed analyses of small regions of interest. Therefore, I devised a co-stable transfection system of DNA fragments of -150 essential region, resulting in linkage of multiple copies of the fragment to the promoter directing the expression of GFP. In this system, We can readily detect the activity of the essential region by flowcytometric analyses without any cell staining. A series of internal deletions of the region revealed a 1.5 kbp core activity in NcoI-SpeI, locating in the middle of the essential region (Appendix, Fig. 4). The nucleotide sequence of the 1.5 kbp fragment revealed several perfect candidate sites for the binding of regulatory factors, including SCL and AML-1. Ianticipate that further dissection of the core element may lead to the identification of a novel mesodermal factor, which we would then examine in greater detail. As soon as the significance of each trans-acting factor on the regulation of GATA-2 is confirmed by further analyses, such factors may also be applied to the in vitro expansion system of hematopoietic stem cells. Less