Cryoultramicrotomy for Subcellular Localization of Molecules

这个项目涉及到仪器的获取、安装和使用，这将使希望之城的研究人员能够在通过电子显微镜成像的组织的薄冰冻切片中对分子进行定量定位。自从电子显微镜用于检查树脂包埋组织的超薄切片以来，关于生命的分子基础的知识已经变得更加详细。为了能够检查亚细胞部位的特定分子，人们开发了一种方法，可以制作冷冻细胞和组织的超薄切片。这样的冷冻切片可以牢固地标记抗体和其他分子，如融合蛋白和衍生的RNA，并用免疫金将这些分子定位到特定的细胞活动区。希望之城的研究人员有强有力的计划，可以在他们的细胞功能研究中利用这种类型的信息。项目包括对突触传递的研究；CD44a及其在形态发生模型中的作用；转基因产物(如bclxl和mdr1)的命运；编码B-G分子的主要组织相容性复合体在病毒感染细胞中的命运；以及p53的定位。正在获得的设备将使在希望之城的这些和其他项目中使用超薄冰冻切片和免疫金标记相结合的细胞定位和共定位的定量分析成为可能。

9970143Abstract This project involves the acquisition, installation and use of instrumentation that will allow investigators at the City of Hope to localize molecules quantitatively in thin cryosections of tissue imaged by electron microscopy. Knowledge of the molecular basis of life has grown considerably more detailed since the inception of electron microcopy for the examination of ultrathin sections of resin embedded tissues. The need to be able to examine particular molecules at subcellular sites has driven the development of a means to make ultrathin sections of frozen cells and tissues. Such cryosections can be labeled robustly with antibodies and other molecules such as fusion proteins and derivitized RNAs, and immunogold to localize these molecules to regions of specialized cellular activity. City of Hope investigators have vigorous programs that can take advantage of this type of information in their studies of cell function. Projects include studies of synaptic transmission; CD44a and its role in a morphogenetic model; the fate of products of transgenes such as Bcl-XL and mdr1; the fate of the major histocompatibility complex encoded B-G molecules in virally-infected cells; and localization of p53. The equipment being acquired will make possible the quantitative analysis of cellular localization and co-localization in these and other projects at the City of Hope using a combination of ultrathin cryosections and immunogold labeling.