REGULATION OF THE CLONED HUMAN BETA-GLOBIN GENE

β地中海贫血小鼠将用于研究外源性调节 转染和移植骨中的小鼠和人类β-珠蛋白基因 骨髓干细胞。 对于这些研究，两个同源菌株 正在开发与 C57BL/6 不同的 β 地中海贫血小鼠 Hbb 基因座上的 DBA/2J 近交伙伴。 血液学参数 这些小鼠的各种骨骼的恢复潜力将被确定 可以评估骨髓疗法。 这些将包括外周血 血液学、红细胞存活、红细胞生成以及红细胞大小 造血组织中的 CFU-S、CFU-C、BFU-E 和 CFU-E 池。 各种 注射的骨髓细胞数量和预处理的组合 接受 X 射线和化学品的接受者将接受调查，以找到 促进长期生存的最佳和/或最实际的条件。 使用这些条件，正常骨髓和β-地中海贫血骨髓的混合物 细胞将被注射到同一受体中以评估相对 正常（作为“基因修复”模型）干细胞的潜力 在宿主骨髓存在的情况下建立功能性移植物 环境。 上述研究将建立用于 研究额外的 β-珠蛋白基因对骨髓的影响 功能。 来自 BALB/c 小鼠和 BALB/c 突变体的骨髓细胞 携带额外的β-珠蛋白基因复合物将被输注致死 照射 BALB/c 小鼠以研究重复片段是否 含有 Hbb 会影响干细胞功能。 CFU-S 的数量和 从培养 24 至 48 小时的骨髓细胞获得的恢复持续时间 正常和转染培养条件下的小时数将与 确定转染本身的效果。 来自脾脏集落的 DNA 来自单个 CFU-S 的衍生品将通过 Southern blotting 进行分析 确定转染的干细胞数量。 的表达水平 长期造血组织中的外源β-珠蛋白基因 幸存者将通过基因特异性蛋白质的定量来确定 或 mRNA 合成。 2 个月和 6 个月幸存者的骨髓将被 重新移植到受到致命辐射的小鼠体内，并从个体中提取DNA 将通过Southern印迹分析脾集落以确定是否 插入出现在完全恢复的骨髓中。 S期特异性药物， 6-硫鸟嘌呤，将用于调查不可分裂Go的比例 循环 CFU-S 细胞，转染并保留外源性 β-珠蛋白基因持续 2 至 6 个月。

Beta-thalassemic mice will be used to study the regulation of exogenous mouse and human beta-globin genes in transfected and transplanted bone marrow stem cells. For these studies, two congenic strains of beta-thalassemic mice are being developed that differ from the C57BL/6 and DBA/2J inbred partners at the Hbb locus. The hematological parameters of these mice will be determined so the restorative potential of various bone marrow therapies can be assessed. These will include peripheral blood hematology, red cell survival, red cell production, and the sizes of the CFU-S, CFU-C, BFU-E and CFU-E pools in the hematopoietic tissues. Various combinations of numbers of marrow cells injected and pretreatment of recipients with X-rays and chemicals will be investigated to find an optimal and/or the most practical condition to promote long-term survival. Using these conditions, mixtures of normal and beta-thalassemic marrow cells will be injected into the same recipient to assess the relative potential of normal (as a model for "genetically repaired") stem cells to establish functional grafts in the presence of the host's marrow and environment. The above studies will establish the protocols to be used to investigate the effects extra beta-globin genes may have on bone marrow function. Bone marrow cells from BALB/c mice and a BALB/c mutant that carries an extra beta-globin gene complex will be transfused into lethally irradiated BALB/c mice to investigate whether the duplicated segment containing Hbb affects stem cell function. The number of CFU-S and duration of recovery obtained from marrow cells incubated for 24 to 48 hours under normal and transfecting culture conditions will be compared to determine the effect of transfection per se. DNA from spleen colonies derived from single CFU-S will be analyzed by Southern blotting to determine the number of stem cells transfected. The level of expression of the exogenous beta-globin genes in hematopoietic tissues of long-term survivors will be determined by quantitation of the gene-specific protein or mRNA synthesis. Bone marrow from 2- and 6-month survivors will be retransplanted into lethally irradiated mice and the DNA from individual spleen colonies will be analyzed by Southern blotting to establish whether the insertions appear in fully restored marrow. A S-phase specific drug, 6-thioguanine, will be used to investigate the proportion of nondividing Go and cycling CFU-S cells that become transfected and retain the exogenous beta-globin genes for 2 to 6 months.