DELETION ANALYSIS OF B GLOBIN YAC TRANSGENES IN ES CELLS

这个建议的目标是确定推定的顺式作用调节序列的作用，对特定阶段的激活和沉默的人γ和β珠蛋白基因。 先前的研究表明，必须保留人类β珠蛋白基因座的天然空间和序列组织，以准确地再现人类个体发育过程中观察到的基因表达模式。 这个小组和其他人已经产生了β珠蛋白YAC转基因小鼠模型，其中在携带野生型基因座的转基因系中观察到的基因表达模式是阶段特异性的并且高度可再现。 然而，YAC的纯化、注射和定位是耗时且技术上困难的。 在这个建议中，我们将开发一个互补的战略，使突变的人β珠蛋白基因座在胚胎干细胞（ES）细胞系来自转基因小鼠携带一个单一的拷贝的人β珠蛋白YAC。 这种方法将允许我们直接比较每个突变的人β珠蛋白转基因在固定的染色体整合位置中的作用。 我们将首先集中在元素接近的γ珠蛋白基因，阶段选择元件（SSE），和假定的5'负调节γ珠蛋白基因。 我们的研究将提供一个更完整的了解在珠蛋白基因调控的顺式元件在发展过程中，并最终可能有助于开发新的遗传和药物治疗地中海贫血和血红蛋白病。

The goals of this proposal are to define the roles of putative cis-acting regulatory sequences on the stage specific activation and silencing of the human gamma and beta globin genes. Previous studies demonstrate that the native spatial and sequence organization of the human beta globin locus must be preserved to accurately recapitulate the pattern of gene expression observed during human ontogeny. This group and others have generated beta globin YAC transgenic mouse models in which the pattern of gene expression observed, in transgenic lines carrying the wild type locus, is stage-specific and highly reproducible. However, the purification, injection and mapping of YACs is time consuming and technically difficult. In this proposal we will develop a complementary strategy by making mutations in the human beta globin locus in embryonic stem (ES) cell lines derived from transgenic mice carrying a single copy of the human beta globin YAC. This approach will allow us to directly compare the effects of each mutated human beta globin transgene in a fixed chromosomal position of integration. We will focus initially on elements proximal to the gamma globin genes, the stage selector element (SSE), and putative 5' negative regulator of gamma globin genes. Our studies will provide a more complete understanding of the cis elements involved in globin gene regulation during development, and may ultimately aid in the development of novel genetic and pharmacolgic therapies for the treatment of thalassemias and hemoglobinopathies.