Identification and functional evaluation of proteins controlling proliferation and differentiation of mammary epithelial cells

乳腺是研究生长、分化和器官发生控制的独特模型。我们计划利用乳腺上皮细胞来鉴定基因，并研究控制其增殖和分化的分子机制。由于癌症的形成与增殖和分化程序的畸变密切相关，因此这些结果也将与乳腺癌研究的进展最为相关。我们最近利用蛋白质组学技术，发现了在乳腺上皮细胞从增殖到完全分化阶段转变过程中存在差异调控的新基因。这些基因可以被分配到控制RNA加工和稳定性，细胞骨架组织和蛋白质折叠和稳定性。我们现在提出(1)改进我们的方法，用一种新的蛋白质组学工具分析亚细胞蛋白部分，(2)使用慢病毒敲低技术在乳原激素应答的乳腺上皮细胞培养模型中定义可解释的候选基因的生理功能，(3)通过移植和转基因原代上皮细胞的乳腺重建实验验证我们的发现。这些实验代表了对乳腺发育中关键基因的全面分析，范围从候选基因鉴定到它们在体外和体内的功能评估。

The mammary gland represents a uniquely suited model for the study of growth, differentiation and the control of organogenesis. We are planning to use mammary epithelial cells to identify the genes and to study the molecular mechanisms that govern their proliferation and differentiation. Since cancer formation is closely linked to aberrations of proliferation and differentiation programs, these results will also be most relevant for progress in breast cancer research. We have recently employed proteomics technology and identified new genes differentially regulated in the transition of mammary epithelial cells from a proliferation to a fully differentiated stage. These genes can be assigned to the control of RNA processing and stability, cytoskeletal organization and protein folding and stability. We now propose (1) to refine our approuch and analyse subcellular protein fractions with a new proteomics tool, (2) define the physiological funtion of interpretable candidate genes in a lactogenic hormone responsive cell culture model of mammary epithelial cells using lentiviral virus based knockdown technology and (3) verify out findings by transplantation and mammary gland reconstitution assays with genetically modified primary epithelial cells. These experiments represent a comprehensive analysis of crucial genes in mammary development and range from candidate gene identification to their functional evaluation in vitro and in vivo.