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

• 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
• 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
• 来源: https://reporter.nih.gov/project-details/6912158
• 关键词: MDCK cell; RNase protection assay; cell nucleus; cell type; flow cytometry; fluorescence microscopy; functional /structural genomics; gene expression; genetically modified animals; green fluorescent proteins; kidney cell; kidney function; laboratory mouse; microarray technology; northern blottings; nucleic acid sequence; polymerase chain reaction; reagent /indicator; renal medulla; single cell analysis; technology /technique development; tissue /cell culture; western blottings

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（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。在患有这些疾病的个体中常见的是血管加压素循环水平升高，血管加压素是一种调节肾脏水排泄的肽激素。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。单个肾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应用最广泛的基因表达全局分析方法之一。传统上，单个基因是在一个实验中研究的，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，目前用于分离组织进行微阵列分析的方法没有解决组织异质性的问题；在有组织的组织中，来自少数细胞亚群的特定信号在来自剩余细胞的一般背景信号中丢失。本研究需要验证的假设是，在细胞特异性启动子的驱动下，核靶向GFP的表达与荧光激活分选相结合，将为特异性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，使我们能够从代表肾脏特定细胞类型的同质细胞核群体中快速纯化RNA，然后通过微阵列进行分析。这些技术基于肾细胞类型特异性启动子的使用，允许将绿色荧光蛋白（GFP）构建物靶向表达到核位置（nGFP）。现在，仅来自肾脏目标细胞的细胞核呈现荧光，允许我们使用流式分选进行纯化。具体目标是：1)在体外验证肾启动子将GFP靶向到肾细胞系的细胞核中；2)在体内发展技术，通过微阵列技术，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选择性标记细胞核亚群的能力，随后通过荧光活化细胞分选（FACS）从组织匀浆中纯化这些细胞核，将提供一种方便、普遍适用和快速的方法来表征任何特定肾细胞类型中的全局基因表达。这项技术将为现有方法提供重大改进，因为目前还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。