The objective is to create a global gene expression atlas of the developing kidney. The central thesis is that a combination of laser capture microdissection and microarrays can be used to efficiently achieve this goal. Microarrays with essentially complete gene representation can be used to rapidly determine the expression levels of every gene in laser capture microdissected elements of the developing kidney. A single experiment, therefore, provides a comprehensive analysis of the gene expression status of one component, and a limited number of experiments examining each structure and substructure can create an atlas.
Specific aim 1 is to use this strategy to produce an atlas of the gene expression profiles of specific domains of the developing mouse kidney. The initial focus will be on the E15.5 kidney, which provides a single time point with multiple stages of nephrogenesis, but earlier time points will also be examined. A combination of structure, lectin staining, immunohistochemistry and transgenic GFP expression will be used to precisely identify specific components and lineages.
Specific aim 2 is to make transgenic mouse tools to promote both gene expression profiling and functional studies of kidney development. We propose to make a series of transgenic mice with specific promoters driving restricted expression of a Cre-GFP cassette. These mice will serve a dual purpose, to allow identification of additional discreet kidney components for Specific Aim 1 and to aid future domain specific gene knockout studies in the developing kidney.
Specific aim 3 is to perform bioinformatics analysis of the microarray data and to make results readily available to the research community. Microarrays produce large gene lists that need to be sifted. Analysis of the complex orchestrations of gene expression defined in Specific Aim 1 will provide deeper insight into the genetic basis of the development of the distinct parts of the nephron.
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