Global Gene Expression Atlas of the Developing Kidney

描述(由申请人提供)：目标是创建一个发育中的肾脏的全球基因表达图谱。中心论点是，激光捕获微解剖和微阵列的结合可以有效地实现这一目标。具有基本完整的基因表示的微阵列可以用来快速确定激光捕获发育中肾脏的显微解剖元件中每个基因的表达水平。因此，一个单独的实验就可以全面分析一种成分的基因表达状态，而检查每个结构和亚结构的有限数量的实验就可以创建一份图谱。具体目标1是利用这一策略制作一份发育中的小鼠肾脏特定区域的基因表达图谱。最初的焦点将放在E15.5肾脏上，它提供了一个具有多个肾脏形成阶段的时间点，但也将检查更早的时间点。将结合结构、凝集素染色、免疫组织化学和转基因GFP表达来准确识别特定的成分和谱系。具体目标2是制造转基因小鼠工具，以促进肾脏发育的基因表达谱和功能研究。我们建议用特定启动子驱动Cre-GFP盒的限制性表达来制备一系列转基因小鼠。这些小鼠将发挥双重作用，为特定的目的1识别额外的谨慎的肾脏成分，并帮助未来在发育中的肾脏中进行特定区域基因敲除研究。具体目标3是对微阵列数据进行生物信息学分析，并使结果易于向研究界提供。微阵列产生需要筛选的大量基因列表。对特定目标1中定义的基因表达的复杂编排的分析将为肾单位不同部分的发育提供更深层次的遗传学基础。

DESCRIPTION (provided by applicant): 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.