GENES EXPRESSED IN STEM CELLS AND DURING CELL CYCLE

这项建议的中心目标是， 在原始细胞中特异性表达的基因的表征 重组鼠造血干细胞。 高浓缩 这些干细胞群将通过标准阳性和阴性纯化， 利用单克隆抗体的阴性选择方法， 荧光染料。 信使RNA（mRNA）将从这些细胞中分离出来 并转化为互补DNA（cDNA_），其将用于构建 使用扩增技术的改进的cDNA文库， 从非常少量的细胞中产生代表性的cDNA文库。 cDNA文库也将被标准化以部分地平衡cDNA文库。 代表丰富和罕见的转录本。 特定于 原始造血干细胞将通过各种展示来鉴定 和捕获方法来区分它们与存在于 控制细胞群，如非造血细胞等 分化的造血祖细胞。 候选人成绩单将 纯化并表征其结构。 cDNA文库也将被 从原始造血祖细胞（FDC-1）群体制备 P1细胞系以及原代扩增的造血细胞）同步化 在造血干细胞的细胞周期的不同阶段， 使用各种cDNA展示和捕获方法分离和表征。 最后，特定的同源异型盒基因产物在调节细胞凋亡中的作用， 将研究原始造血祖细胞的基因表达 使用快速高振幅基因诱导系统。 通过这些方法， 预期可以鉴定多种新的基因产物， 包括细胞表面标志物、受体、转录因子和其它 早期反应基因产物。 这种新的基因产物可以用于 作为各种策略和技术中的试剂， 富集的，甚至是纯的原始造血干细胞， 用于细胞生物学研究、移植的定向祖细胞 和基因转移/治疗程序。

This proposal has as its central objective, the isolation and characterization of genes that are specifically expressed in primitive reconstituting murine hematopoietic stem cells. Highly enriched populations of such stem cells will be purified by standard positive and negative selection procedures utilizing monoclonal antibodies and fluorescent dyes. Messenger RNA (mRNA) will be isolated from these cells and converted into complementary DNA (cDNA_) that will be used to construct cDNA libraries using modifications of amplification techniques capable of generating representative cDNA libraries from very small numbers of cells. The cDNA libraries will also be normalized to partially equalize the representation of abundant and rare transcripts. Transcripts specific for primitive hematopoietic stem cells will be identified by various display and capture methods to differentiate them from transcripts present in control cell populations such as non hematopoietic cells and more differentiated hematopoietic progenitor cells. Candidate transcripts will be purified and their structure characterized. cDNA libraries will also be prepared from populations of primitive hematopoietic progenitor cells (FDC- P1 cell line as well as primary explanted hematopoietic cells) synchronized at different stages of the cell cycle in hematopoietic stem cells will be isolated and characterized using various cDNA display and capture methods. Finally, the role of specific homeobox gene products ina the regulation of gene expression of primitive hematopoietic progenitor cells will be studied using a rapid high amplitude gene induciton system. By these methods, it is anticipated that a variety of novel gene products can be identified, including cell surface markers, receptors, transcriptions factors and other early response gene products. Such novel gene products could then be used as reagents in various strategies and techniques to obtain more highly enriched, even pure, primitive hematopoietic stem cells as well as committed progenitor cells for cellular biological studies, transplantation and gene transfer/therapy procedures.