Nuclear architecture of gene expression: 3D topology of transcribed and silenced genes in mouse embryonic stem cells and cells differentiated from them

越来越多的证据表明核组织在调控基因组中起着重要作用。本次申请的研究将从三个维度探讨基因在固定细胞和活细胞分化过程中的表达背景及其在细胞核中的定位。我们将稳定地转染小鼠胚胎干（ES-）细胞，其中含有用于细胞类型特异性正确调控的基因座控制区（LCR）和绿色荧光蛋白（GFP）标签。后者允许显微检测活细胞和固定细胞中的整合基因。我们将在沉默的es细胞中观察转基因，在从这些es细胞分化出来的细胞中观察转基因是否转录。我们将使用b-珠蛋白基因和鸡溶菌酶基因作为转基因。两者在分子水平上都有广泛的特征。因此，我们将能够整合微观和分子观察。内源基因的对照实验将通过多色荧光原位杂交（FISH）进行。

Increasing evidence points to an important role of nuclear organization in regulating the genome. The study applied for will investigate the context of the expression of genes and their localization in the cell nucleus in three dimensions in both, fixed and living cells during differentiation. We will do so by stably transfecting mouse embryonic stem (ES-) cells with transgenes containing a Locus Control Region (LCR) for cell type specific correct regulation plus a tag for a Green Fluorescent Protein (GFP) label. The latter allows microscopic detection of the integrated gene in living and fixed cells. We will observe the transgenes in ES-cells where they are silenced and in cells differentiated from these ES-cells were the transgenes are either transcribed or not transcribed. We will use the b-Globin gene and the chicken lysozyme gene as transgenes. Both are extensively characterized on the molecular level. Thus we will be able to integrate microscopical and molecular observations. Control experiments with the endogenous genes will be performed by multi-color Fluorescence in situ Hybridization (FISH).