DELETION ANALYSIS OF B GLOBIN YAC TRANSGENES IN ES CELLS

本提案的目的是确定假定的顺式调控序列在人类γ和β珠蛋白基因的阶段特异性激活和沉默中的作用。先前的研究表明，必须保留人类β -珠蛋白位点的固有空间和序列组织，才能准确地概括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建立了β珠蛋白YAC转基因小鼠模型，在携带野生型位点的转基因系中观察到的基因表达模式具有阶段特异性和高度可重复性。然而，YACs的纯化、注入和映射是耗时且技术困难的。在本研究中，我们将开发一种补充策略，即在胚胎干（ES）细胞系中对携带人类β -珠蛋白YAC单拷贝的转基因小鼠的人β -珠蛋白位点进行突变。这种方法将使我们能够直接比较每个突变的人类球蛋白转基因在固定染色体整合位置的影响。我们将首先关注接近γ珠蛋白基因的元件，阶段选择元件（SSE）和假定的γ珠蛋白基因的5'负调节因子。我们的研究将对发育过程中参与珠蛋白基因调控的顺式元件提供更全面的了解，并可能最终有助于开发新的遗传和药物疗法来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.