Center for in Toto Genomic Analysis of Vertebrate Development

描述（由申请人提供）：基因组科学卓越中心（CEGS）汇集了一个多学科的研究人员小组，以开发创新技术，目标是对每一个发育重要的脊椎动物基因进行成像和突变。新的“全成像”工具使人们有可能使用一个系统为基础的方法来分析基因功能发育脊椎动物胚胎在真实的时间和空间。 这些工具可以以系统、高通量和定量的方式分析体内数据。 将全成像与新型基因陷阱相结合，可以快速筛选发育相关的表达模式，然后能够立即诱变感兴趣的基因。最初，由于斑马鱼胚胎的透明度和获得快速反馈的能力，关键技术将在斑马鱼胚胎中开发和测试。一旦得到验证，这些技术将被应用于一个鸟类胚胎，由于几个优点，包括可访问性和类似于人类胚胎发生。最后，为了监测正常和突变胚胎中基因表达的变化，我们将开发原位杂交新技术，以敏感和潜在的定量方式同时分析多个标记基因。我们的目标是结合联合收割机在全基因组范围内对基因表达的真实的时间分析，以及使感兴趣的基因突变的能力，并检查由于基因丢失而导致的基因表达的全局改变。大部分价值将来自新的和广泛适用的技术的发展。然而，与典型的技术开发赠款相比，至少会有两种脊椎动物系统（斑马鱼和鸟类）的实验成果。将努力实现以下目标： 具体目标1：报告基因表达的实时“整体”图像分析。 具体目标二：使用FlipTrap方法进行基因捕获，对发育中的脊椎动物胚胎的基因功能进行全面的时空分析。 具体目标3：设计定量，多重“杂交链反应”（HCR）放大器，用于具有主动背景抑制的体内成像。 具体目标4：数据分析和数据集的整合，以产生“数字”鱼和“数字”鸟。这些技术和由此产生的地图集将通过电子出版物广泛提供。

DESCRIPTION (provided by applicant): This Center of Excellence in Genomic Science (CEGS) assembles a multidisciplinary group of investigators to develop innovative technologies with the goal of imaging and mutating every developmentally important vertebrate gene. Novel "in toto imaging" tools make it possible to use a systems-based approach for analysis of gene function in developing vertebrate embryos in real time and space. These tools can digitize in vivo data in a systematic, high-throughput, and quantitative fashion. Combining in toto imaging with novel gene traps permits a means to rapidly screen for developmentally relevant expression patterns, followed by the ability to immediately mutagenize genes of interest. Initially, key technologies will be developed and tested in the zebrafish embryo due to its transparency and the ability to obtain rapid feedback. Once validated, these techniques will be applied to an amniote, the avian embryo, due to several advantages including accessibility and similarity to human embryogenesis. Finally, to monitor alterations in gene expression in normal and mutant embryos, we will develop new techniques for in situ hybridization that permit simultaneous analysis of multiple marker genes in a sensitive and potentially quantitative manner. Our goal is to combine real time analysis of gene expression on a genome-wide scale coupled with the ability to mutate genes of interest and examine global alterations in gene expression as a result of gene loss. Much of the value will come from the development of new and broadly applicable technologies. In contrast to a typical technology development grant, however, there will be experimental fruit emerging from at least two vertebrate systems (zebrafish and avian). The following aims will be pursued: Specific Aim 1: Real-time "in toto" image analysis of reporter gene expression. Specific Aim 2: Comprehensive spatiotemporal analysis of gene function of the developing vertebrate embryo using the FlipTrap approach for gene trapping. Specific Aim 3: Design of quantitative, multiplexed 'hybridization chain reaction' (HCR) amplifiers for in vivo imaging with active background suppression. Specific Aim 4: Data analysis and integration of data sets to produce a "digital" fish and a "digital" bird. The technologies and the resulting atlases will be made broadly available via electronic publication.