Targeting Nuclei for Renal Cell Specific Gene Analysis

描述(申请人提供)：糖尿病、充血性心力衰竭、肝硬变和肾病综合症都是与肾脏在处理盐和水方面的缺陷有关的疾病。在患有这些疾病的人中，常见的是循环中的加压素水平升高，这是一种调节肾脏水分排泄的多肽激素。通常只有一种特定细胞类型的功能变化是疾病状态发展的基础。然而。单个肾脏细胞类型的基因表达研究经常受到分离和提取感兴趣细胞的方法的阻碍。DNA微阵列技术已迅速发展成为全球基因表达分析中应用最广泛的方法之一。传统上，在单个实验中研究单个基因，现在我们有能力在一个对照实验中同时研究数千个基因的调控。然而，目前用于分离用于微阵列分析的组织的方法没有解决组织异质性的问题；来自有组织组织内的少数细胞亚群的特定信号在来自剩余细胞的信号的一般背景中丢失。这项研究的假设是，在细胞特异性启动子的驱动下，核靶向GFP的表达，加上荧光激活的分选，将为特定类型的肾脏细胞的功能基因组分析提供一种方法学。我们将开发新的试剂和技术，使我们能够快速纯化，然后通过微阵列从代表肾脏特定细胞类型的同质核群体中分析RNA。这些技术是基于使用肾细胞类型特异性启动子来允许将绿色荧光蛋白(GFP)构建物靶向表达到核位置(NGFP)。只有肾脏靶细胞的细胞核现在呈现荧光，使我们能够使用流动分选来纯化它们。其具体目的是1)体外验证肾细胞系中GFP靶向细胞核的肾启动子，2)发展体内技术，利用微阵列分析表达nGFP的转基因小鼠肾细胞特异性核的基因表达。在体内选择性地用GFP标记细胞核亚群，并随后通过荧光激活细胞分选(FACS)从组织匀浆中纯化这些核的能力，将提供一种方便、普遍适用的快速手段来表征任何特定类型的肾脏细胞中的全球基因表达。这项技术将提供比现有方法显著的改进，因为没有其他技术可以全局分析体内特定细胞类型的基因表达。

DESCRIPTION (provided by applicant): Diabetes, congestive heart failure, cirrhosis of the liver and nephrotic syndrome are all diseases which have been associated with defects in the handling of salt and water by the kidney. Common in individuals with these disorders are elevated circulating levels of vasopressin, the peptide hormone that regulates renal water excretion. It is often the change in function of only one specific cell type that underlies the development of a disease state. However. gene expression studies of individual renal cell types are often hampered by methods to isolate and extract the cells of interest. DNA microarray technology has rapidly developed as one of the most widely used methods for global analysis of gene expression. Traditionally, a single gene was studied in a single experiment, now we have the ability to study the regulation of thousands of genes simultaneously, in a controlled experiment. However, current methods used to isolate tissues for microarray analysis do not address the problem of tissue heterogeneity; specific signals from minor subpopulations of cells within organized tissues are lost within the general background of signals from the remaining cells. The hypothesis to be tested in this study is that expression of nuclear targeted GFP, driven by cell specific promoters, coupled to fluorescence activated sorting, will provide a methodology for functional genomic analysis of specific renal cell types. We will develop new reagents and techniques that will enable us to rapidly purify, and then analyze by microarray, RNA from a homogeneous population of nuclei representing specific cell types in the kidney. These techniques are based on the use of renal cell type specific promoters to allow targeted expression of Green Fluorescent Protein (GFP) constructs to a nuclear location (nGFP). Nuclei from only targeted cells in the kidney are now rendered fluorescent, allowing us to use flow sorting for their purification. The specific aims are 1) Validation in vitro of renal promoters for targeting GFP to the nucleus in renal cell lines and 2) Development of techniques in vivo, for gene expression analysis, by microarray, of renal cell specific nuclei from nGFP expressing transgenic mice. The ability to selectively label subsets of nuclei with GFP in vivo, and subsequently to purify these nuclei from tissue homogenates via fluorescent activation cell sorting (FACS) will provide a convenient, universally applicable, and rapid means to characterize global gene expression within any particular renal cell type. This technology will provide significant improvement over existing approaches, since no other techniques exist to globally analyze gene expression within specific cell types in vivo.