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.
|Effective start/end date||7/15/03 → 6/30/06|
- National Institutes of Health: $16,347.00
- National Institutes of Health: $143,860.00
- National Institutes of Health: $20,219.00
- National Institutes of Health: $151,000.00