CHARACTERIZATION OF A GENE STIMULATIVE STEM CELL RENEWAL

描述：(改编自研究人员摘要)造血细胞 BL3株携带重排的N2-IL-3逆转录病毒基因组。这些细胞是 来自一只患有骨髓增生性疾病和淋巴的小鼠 骨髓细胞感染逆转录病毒后的结节增大 含有新霉素抗性和IL-3基因的载体。这些细胞 类似于未成熟的原始细胞和部分新霉素的缺失 抗性基因、IL-3基因和3‘LTR基因解释了 重新编排。因此，重新排列的病毒基因组也提供了一种 BL3细胞的基因标签。有了这个标签，首席调查员 研究BL3细胞能否重建受致死性照射的小鼠。脱氧核糖核酸 对临床正常受者的器官进行Southern印迹分析 分析。LTR-NEO/NEO特定带型与 亲本BL3细胞系在胸腺、脾、骨髓和 肺，但不在肝脏或肾脏。骨髓甲基纤维素法 来自受者的细胞显示存在非因子依赖的 由分化的粒细胞和巨噬细胞组成的集落。这些 数据表明永生化的BL3细胞可以在体内分化 并产生功能成熟的淋巴和造血细胞 髓系血统。确定BL3细胞是否具有长期 在重新填充潜力时，首席调查员重复了 7个月后对小鼠进行重建实验和分析。他 表明只有100个BL3细胞就可以拯救一个致命的- 受辐射的接受者。分离脾细胞并对其进行浓缩 具有Percoll梯度的淋巴样细胞或髓样细胞。此外，细胞也被 用PHA或WEHI-3条件培养液刺激。特定于BL3的重新排列 在每个浓缩的细胞群体中都存在条带。通过一系列 分子遗传分析，首席调查员已经开始 鉴定导致BL3细胞永生化的基因。北方 对这些细胞的印迹RNA分析显示，存在一种新的 大约3 kb。他构建了Okyama-Borg CDNA表达文库 从BL3细胞的RNA中获得了一个2.75kb的克隆 插入与LTR-NEO探头类似的杂交。克隆这一点 杂交基因正在进行中，目的是它可能与 这项提议使BL3细胞永生。首席调查员 计划更全面地鉴定BL3细胞以确定基因 为了使BL3细胞永生化，并检查基因组 一旦确定了这种基因的组织结构。

DESCRIPTION: (Adapted from investigator's abstract) The hematopoietic cell line, BL3, carries a rearranged N2-IL-3 retroviral genome. The cells were derived from a mouse which developed a myeloproliferative disease and lymph node enlargement after its marrow cells were infected with a retroviral vector containing the neomycin resistance and the IL-3 genes. The cells resemble immature blast cells and the deletion of a portion of the neomycin resistance gene, the IL-3 gene, and the 3' LTR accounts for the rearrangement. The rearranged viral genome, therefore, also provides a genetic tag for BL3 cells. With this tag, the Principal Investigator has studied whether BL3 cells could reconstitute lethally irradiated mice. DNA from organs of clinically normal recipients were analyzed by Southern blot analysis. An LTR-neo/neo specific band pattern identical to that in the parental BL3 cell line was observed in the thymus, spleen, marrow, and lung, but not in the liver or kidney. Methylcellulose assays of marrow cells from the recipients revealed the presence of factor-independent colonies composed of differentiated granulocyte and macrophage cells. These data suggested that the immortalized BL3 cells could differentiate in vivo and give rise to functional and mature hematopoietic cells of lymphoid and myeloid lineages. To determine whether BL3 cells had long term repopulating potential, the Principal Investigator repeated the reconstitution experiments and analyzed the mice 7 months later. He demonstrated that as few as 100 BL3 cells could rescue a lethally- irradiated recipient. Spleen cells were isolated and were enriched for lymphoid or myeloid cells with a percoll gradient. Also, cells were stimulated with PHA or WEHI-3-conditioned media. BL3-specific rearranged bands were present in each enriched cell population. With a series of molecular genetic analyses, the Principal Investigator has begun to identify the gene accounting for the immortalization of BL3 cells. Northern blot RNA analysis of these cells revealed the presence of a novel MRNA of approximately 3 kb. He constructed an Okyama-Borg CDNA expression library from RNA of BL3 cells and obtained one clone that contained a 2.75 kb insert which similarly hybridized with the LTR-neo probe. Cloning of this hybrid gene is underway with the intent that it may relate to the immortalization of BL3 cells with the proposal. The Principal Investigator plans to characterize BL3 cells more fully to identify the gene accounting for the immortalization of BL3 cells, and to examine the genomic organization of this gene once identified.