GENES EXPRESSED IN STEM CELLS AND DURING CELL CYCLE

这项提议的中心目标是孤立和 原始细胞中特异表达的基因的特征 重建小鼠造血干细胞。高度浓缩 这样的干细胞群体将通过标准的阳性和 使用单抗和阴性选择程序 荧光染料。将从这些细胞中分离出信使RNA(MRNAs) 并转化为互补DNA(CDNA1)，将用于构建 使用改进的扩增技术的cDNA文库能够 从极少量的细胞中生成具有代表性的cDNA库。 CDNA文库也将被归一化，以部分平衡 丰富而稀有的抄本。特定于 原始造血干细胞将通过不同的显示方式进行鉴定 和捕获方法，以将它们与 控制细胞群体，如非造血细胞和更多 分化的造血祖细胞。候选人的成绩单将 对其进行纯化，并表征其结构。CDNA库也将被 从原始造血祖细胞(FDC- P1细胞系以及原代移植的造血细胞)同步 在细胞周期的不同阶段，造血干细胞将 用不同的cDNA展示和捕获方法进行分离和鉴定。 最后，特定的同源异型盒基因产物在调节 将研究原始造血祖细胞的基因表达 使用快速、高幅度的基因诱导系统。通过这些方法，它 预计可以鉴定出各种新的基因产物， 包括细胞表面标志物、受体、转录因子等 早期反应基因产物。这样的新基因产品就可以用来 作为试剂在各种策略和技术中获得更高的 浓缩的，甚至是纯净的原始造血干细胞以及 致力于细胞生物学研究、移植的祖细胞 以及基因转移/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.