Targeting Nuclei for Renal Cell Specific Gene Analysis

靶向细胞核进行肾细胞特异性基因分析

基本信息

批准号：
6912158
负责人：
HEDDWEN L BROOKS
金额：
$ 2.02万
依托单位：
UNIVERSITY OF ARIZONA
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-07-15 至 2006-06-30
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：糖尿病、充血性心力衰竭、肝硬化和肾病综合征都是与肾脏处理盐和水的缺陷有关的疾病。患有这些疾病的个体常见的是加压素循环水平升高，加压素是调节肾水排泄的肽激素。通常，只有一种特定细胞类型的功能变化才是疾病状态发展的基础。然而。个别肾细胞类型的基因表达研究常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使用最广泛的方法之一。传统上，在单个实验中研究单个基因，现在我们有能力在受控实验中同时研究数千个基因的调控。然而，目前用于分离组织进行微阵列分析的方法并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其余细胞的信号的一般背景中丢失。本研究要测试的假设是，由细胞特异性启动子驱动的核靶向 GFP 的表达，与荧光激活分选相结合，将为特定肾细胞类型的功能基因组分析提供一种方法。我们将开发新的试剂和技术，使我们能够快速纯化代表肾脏中特定细胞类型的同质细胞核群体的 RNA，然后通过微阵列进行分析。这些技术基于使用肾细胞类型特异性启动子，以允许绿色荧光蛋白 (GFP) 构建体靶向表达到核位置 (nGFP)。仅来自肾脏中的目标细胞的细胞核现在呈现荧光，使我们能够使用流式分选来纯化它们。具体目标是 1) 体外验证肾脏启动子将 GFP 靶向肾细胞系的细胞核，以及 2) 开发体内技术，通过微阵列对表达 nGFP 的转基因小鼠的肾细胞特异性细胞核进行基因表达分析。在体内用 GFP 选择性标记细胞核子集，然后通过荧光激活细胞分选 (FACS) 从组织匀浆中纯化这些细胞核的能力将提供一种方便、普遍适用且快速的方法来表征任何特定肾脏内的全局基因表达。细胞类型。该技术将比现有方法提供显着改进，因为没有其他技术可以全面分析体内特定细胞类型内的基因表达。