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.

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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