Center for in Toto Genomic Analysis of Vertebrate Development

描述（由申请人提供）：这个基因组科学卓越中心（CEGS）汇集了一个多学科的研究小组，以开发创新技术为目标，成像和突变每一个重要的脊椎动物基因。新颖的“体内成像”工具使得使用基于系统的方法实时和空间地分析发育中的脊椎动物胚胎的基因功能成为可能。这些工具可以以系统、高通量和定量的方式对体内数据进行数字化。将toto成像与新型基因陷阱相结合，可以快速筛选与发育相关的表达模式，然后立即诱变感兴趣的基因。最初，关键技术将在斑马鱼胚胎中开发和测试，因为它的透明度和获得快速反馈的能力。一旦得到验证，这些技术将应用于羊膜，鸟类胚胎，由于几个优点，包括可获得性和与人类胚胎发生的相似性。最后，为了监测正常和突变胚胎中基因表达的变化，我们将开发新的原位杂交技术，允许以敏感和潜在的定量方式同时分析多个标记基因。我们的目标是结合全基因组范围内基因表达的实时分析，以及突变感兴趣的基因的能力，并检查由于基因丢失而导致的基因表达的全局变化。大部分价值将来自新技术和广泛应用技术的发展。然而，与典型的技术开发拨款不同，至少有两种脊椎动物系统（斑马鱼和鸟类）将产生实验成果。将实现下列目标：

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.