MAINTENANCE OF GENE EXPRESSION IN THE RED CELL LINEAGE

描述（改编自本申请）：在红细胞分化过程中， 基因组逐渐包装成异染色质， 沉默确定LCR和增强剂预防的机制 沉默，LCR 5'HS2抑制转基因沉默的能力被证明是有效的。 在K562细胞中的异位基因组位点进行测试。活性转基因含有 当沉默时，完整增强子远离着丝粒异染色质定位 转基因更接近着丝粒异染色质。突变 削弱增强子抑制沉默的能力， 转基因，即使在活性状态下，保持接近着丝粒。 细胞中基因表达的激活和维持的分子基础 红系细胞将通过以下定义：1）分离转基因整合 位点以检查转基因和侧翼区之间的相互作用。（二） 确定与活化相关的顺式序列和反式因子， 沉默的定义基因组位点，重点是核因子相关 活跃和不活跃的核隔间。3)导出新的K562集成 无需选择表达即可靶向的位点 关于LCR和染色质绝缘体性质的基本问题， 和4）开发小鼠模型以研究增强子在红细胞增殖过程中的功能， 体内成熟。这将有助于研究 红系增强子抑制小鼠红系成熟期间的沉默， 确定体内沉默的抑制是否涉及 在细胞核内转基因。

DESCRIPTION (adapted from the application): During red cell differentiation the genome is progressively packaged into heterochromatin and gene expression silenced. To define the mechanisms by which LCRs and enhancers prevent silencing, the ability of LCR 5'HS2 to suppress silencing of transgenes was tested at ectopic genomic sites in K562 cells. Active transgenes containing an intact enhancer localize away from centromeric heterochromatin while silenced transgenes are in closer proximity to centromeric heterochromatin. Mutations that impair the ability of the enhancer to suppress silencing cause the transgene, even when in an active state, to remain in proximity to centromeres. The molecular basis for activation and maintenance of gene expression in erythroid cells will be defined by: 1) isolation of the transgene integration sites to examine interactions between the transgene and flanking region. 2) determining the cis sequences and trans factors associated with activation and silencing of defined genomic loci, focusing on nuclear factors associated with active and inactive nuclear compartments. 3) deriving new K562 integration sites that can be targeted without selection for expression to address fundamental questions regarding the nature of LCRs and chromatin insulators, and 4) developing a mouse model to study enhancer function during red cell maturation in vivo. This will allow investigation of the mechanism by which erythroid enhancers suppress silencing during erythroid maturation in mice and determine if suppression of silencing in vivo involves relocalization of the transgene within the nucleus.