REGULATION OF THE CLONED HUMAN BETA-GLOBIN GENE

克隆人β-珠蛋白基因的调控

• 批准号: 3352589
• 负责人: RAYMOND A POPP
• 金额: $ 10.48万
• 依托单位: LOCKHEED MARTIN ENERGY SYSTEMS, INC.
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 1991-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3352589
• 关键词: beta globulin; blood cell count; bone marrow transplantation; erythroid stem cell; gene therapy; genetic manipulation; genetic strain; hematopoietic stem cells; immunosuppressive; myeloid stem cell; radiation immunosuppression; thalassemia

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.

β-地中海贫血小鼠将被用于研究外源性 小鼠和人β-珠蛋白基因在转基因和移植骨中的表达 骨髓干细胞。在这些研究中，两个同源菌株 正在培育不同于C57BL/6和C57BL/6的β地中海贫血小鼠 DBA/2J是Hbb基因座上的近交系。血液学指标的变化 将测定这些小鼠的各种骨骼的修复潜力 骨髓疗法是可以评估的。这将包括外周血 血液学，红细胞存活率，红细胞生成，以及 造血组织中CFU-S、CFU-C、BFU-E和CFU-E池。五花八门 注射的骨髓细胞数量和预处理的组合 接受X光和化学物质治疗的人将接受调查，以发现 最佳和/或最实际的条件，以促进长期生存。 在这些条件下，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者体内以评估亲属 正常(作为“基因修复”的模型)干细胞的潜能 在宿主骨髓存在的情况下建立功能性移植物 环境。上述研究将确定用于以下目的的方案 研究额外的β-珠蛋白基因可能对骨髓的影响 功能。来自BALB/c小鼠的骨髓细胞和BALB/c突变体 携带额外的β-珠蛋白基因复合体将被注入致死的 照射后的BALB/c小鼠研究复制片段是否 含有Hbb会影响干细胞功能。CFU-S和 培养24至48天的骨髓细胞的恢复时间 在正常和转基因培养条件下的培养时间将与 确定转染剂本身的效果。从脾集落中提取DNA 来自单个CFU的S将通过Southern blotting进行分析以 确定干细胞的转染量。的表达水平 外源性β-珠蛋白基因在远期造血组织中的表达 幸存者将通过对基因特异性蛋白的量化来确定 或信使核糖核酸的合成。2个月和6个月幸存者的骨髓将被 再移植到致死照射的小鼠体内和来自个体的DNA 将通过Southern blotting分析脾克隆以确定 这些植入物出现在完全修复的骨髓中。S期特效药， 6-硫代鸟嘌呤，将用于研究不分割GO的比例 和循环CFU-S细胞，成为转染并保留外源 持续2至6个月的β-珠蛋白基因。