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，核组织在调节基因组中发挥着重要作用。申请的研究将调查基因表达的背景及其在细胞核中的三维定位，在分化过程中固定和活细胞。我们将通过用含有用于细胞类型特异性正确调节的基因座控制区（LCR）和用于绿色荧光蛋白（GFP）标记的标签的转基因稳定转染小鼠胚胎干（ES-）细胞来实现这一目标。后者允许在活细胞和固定细胞中显微镜检测整合的基因。我们将观察ES细胞中的转基因，其中它们被沉默，并且在从这些ES细胞分化的细胞中，转基因被转录或不转录。我们将使用b-珠蛋白基因和鸡溶菌酶基因作为转基因。两者都在分子水平上被广泛表征。因此，我们将能够整合显微镜和分子观察。将通过多色荧光原位杂交（FISH）进行内源性基因的对照实验。

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).