CHARACTERIZATION OF A GENE STIMULATING STEM CELL RENEWAL

描述：（改编自研究者摘要）造血细胞 BL 3系携带重排的N2-IL-3逆转录病毒基因组。 将细胞 来源于患有骨髓增生性疾病和淋巴瘤的小鼠， 在它的骨髓细胞被逆转录病毒感染后， 含有新霉素抗性和IL-3基因的载体。细胞 类似于未成熟的原始细胞，并且缺失了新霉素的一部分， 抗性基因、IL-3基因和3' LTR解释了 重排 因此，重排的病毒基因组还提供了一种 BL 3细胞的基因标签。 有了这个标签，首席研究员 研究BL 3细胞是否能重建致死辐射的小鼠。DNA 用Southern印迹法分析临床正常受者的器官 分析. LTR-neo/neo特异性条带模式与 在胸腺、脾、骨髓和胸腺中观察到亲本BL 3细胞系， 肺，但不是在肝脏或肾脏。骨髓甲基纤维素测定 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 结果表明，永生化BL 3细胞可在体内分化 并产生功能性和成熟的淋巴造血细胞， 骨髓谱系 为了确定BL 3细胞是否具有长期的 重新填充潜力，主要研究者重复了 重组实验，并在7个月后分析小鼠。 他 证明了只要100个BL 3细胞就可以拯救一个致命的- 辐射受体 分离脾细胞，并富集用于 淋巴样或髓样细胞，具有梯度。 此外，细胞 用PHA或WEHI-3条件培养基刺激。 BL 3特异性重排 在每个富集的细胞群中存在条带。 用一系列 分子遗传学分析，首席研究员已经开始 确定BL 3细胞永生化的基因。北方 这些细胞的印迹RNA分析揭示了一种新的mRNA的存在， 大约3 KB。 他构建了Okyama-Borg cDNA表达文库 从BL 3细胞的RNA中，获得了一个含有2.75 kb的 与LTR-neo探针类似杂交的插入物。 克隆这个 杂交基因正在进行中，目的是它可能与 BL 3细胞永生化的建议。 主要研究者 计划更全面地描述BL 3细胞，以确定基因计数 BL 3细胞的永生化，并检查基因组 这个基因一旦被发现。

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.