REGULATION OF THE CLONED HUMAN BETA-GLOBIN GENE

β-地中海贫血小鼠将用于研究外源性 转染和移植骨中的小鼠和人β-珠蛋白基因 骨髓干细胞 对于这些研究，两个同源菌株的 正在开发的β-地中海贫血小鼠不同于C57 BL/6， DBA/2 J近交伴侣在Hbb基因座。 血液学参数 这些小鼠将被确定， 可以评估骨髓疗法。 这些将包括外周血 血液学、红细胞存活率、红细胞生成和红细胞大小 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射线和化学品的人将接受调查， 最佳和/或最实用的条件，以促进长期生存。 使用这些条件，正常和β地中海贫血骨髓的混合物 细胞将被注射到同一个受体中， 正常干细胞（作为“基因修复”的模型）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以下目的的方案： 研究额外的β-珠蛋白基因对骨髓的影响 功能 来自BALB/c小鼠的骨髓细胞和BALB/c突变体， 携带一个额外的β-珠蛋白基因复合体将被注入致命的 照射BALB/c小鼠，以研究是否重复段 含有Hbb影响干细胞功能。 CFU-S数量和 从培养24 - 48小时的骨髓细胞中获得的恢复持续时间 将在正常和离心培养条件下的培养时间与 确定转染本身的效果。 脾菌落DNA 将通过Southern印迹法分析源自单个CFU-S的细胞， 确定转染的干细胞的数量。 的表达水平 外源性β-珠蛋白基因在长期造血组织中的表达 将通过定量基因特异性蛋白来确定存活者 或mRNA合成。 将采集2个月和6个月存活者的骨髓， 再次移植到致命辐射的小鼠和DNA从个人 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。 一种S期特异性药物， 6-硫鸟嘌呤，将用于研究非分裂Go的比例 以及循环CFU-S细胞，其被转染并保留外源性 β-珠蛋白基因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.