TRANSGENIC MODLES AND GENETIC MODIFIERS OF SICKLE CELL DISEASE

本提案的第一个目标是生产改良的转基因小鼠 镰状细胞病（SCD）的模型。导致血管- 闭塞、疼痛危象和SCD的其他病理生理学方面是 复杂且不为人知。动物模型对于体内研究可能是有价值的 急性和慢性血管闭塞过程的研究，并用于测试 药物或基因疗法。表达人HbS的转基因系将是 与α和β珠蛋白基因缺失的小鼠杂交， 产生表达高达100%人HbS的后代。这些老鼠将 表型特征为新SCD模型。第二个目标是 使用技术鉴定镰状细胞病的新遗传修饰剂 来绘制老鼠的基因图谱。虽然主要的遗传决定因素 SCD是β 6Val突变，在突变中存在相当大的变异性。 SS纯合子中疾病的表型表达，这可能是 部分原因是遗传修饰。这种基因的鉴定 修饰物可以提出新的治疗策略，并提供 个体SCD患者的预后指标。虽然这将是 通过人类遗传学研究很难识别这些基因，最近 小鼠遗传学的发展允许修饰基因的快速作图。 将SAD-1转基因SCD模型首先培育成不同的近交系 背景以鉴定其中相同转基因具有 数量上不同的表型效应修饰基因座的数目 将从亲本、F1和N2回交分析中估计 代为了绘制修饰基因座，将N2动物的大池（a large pool of N2 animals） 表型表征，并使用多态性PCR标记进行基因分型， 跨越基因组。预测修饰子附近的候选基因定位 将检查两个菌株之间的等位基因差异， 鉴定潜在修饰基因。除了这一全球办法之外， 红细胞-内皮细胞相互作用的预测修饰剂，血管内皮细胞， 将通过SAD-1小鼠之间的杂交检测维勒布兰德因子（vWF） 和近交系RIIIS/J小鼠，其表达低水平的vWF。

The first goal of this proposal is to produce improved transgenic mouse models of sickle cell disease (SCD). The processes leading to vaso- occlusion, painful crisis and other pathophysiological aspects of SCD are complex and poorly understood. Animal models may be valuable for in vivo studies of acute and chronic vaso-occlusive processes, and for testing drug or gene therapies. A transgenic line expressing human HbS will be crossed with mice whose alpha and beta globin genes are deleted, to generate offspring expressing up to 100% human HbS. These mice will be phenotypically characterized as new SCD models. The second goal is to identify novel genetic modifiers of sickle cell disease, using techniques for genetic mapping the mouse. While the primary genetic determinant of SCD is the beta6Val mutation, there is considerable variability in the phenotypic expression of the disease among SS homozygotes, which may be due in part to genetic modifiers. The identification of such genetic modifiers could suggest new strategies for treatment, and provide prognostic indicators for individual SCD patients. While it would be difficult to identify such genes through human genetic studies, recent developments in mouse genetics permit the rapid mapping of modifier genes. The SAD-1 transgenic SCD model first will be bred into different inbred backgrounds to identify two strains in which the same transgene has quantitatively different phenotypic effects. The number of modifier loci will be estimated from an analysis of parental, F1 and N2 backcross generations. To map the modifier loci, a large pool of N2 animals will phenotypically characterized, and genotyped using polymorphic PCR markers, spanning the genome. Candidate genes mapping near the predicted modifier loci will be examined for allelic differences between the two strains, to identify potential modifier genes. In addition to this global approach, the role of a predicted modifier of red cell-endothelial interactions, von Willebrand factor (vWF), will be tested through crosses between SAD-1 mice and inbred RIIIS/J mice, which express lowered levels of vWF.