Generation and in vivo validation of cis-regulatory maps in eukaryotic genomes

描述（由申请人提供）：ENCODE项目的初始阶段导致了几种高通量策略的开发和建立，用于快速鉴定人类基因组中预测的功能元件。目前缺乏足够的系统来实验验证这些预测，特别是可以测试作为转录沉默子和边界元件的元件的方法。我们建议建立一个体外和体内平台，以便有效地测试人类基因组中预测的增强子、沉默子和绝缘子。我们将通过同源重组以位点特异性方式整合报告载体，以4种人类细胞系和小鼠胚胎干细胞为目标。通过使用各种载体设计，我们将开发可用于测试基因组序列转录激活特性以及沉默和边界特性的细胞系，独立于变异和多拷贝整合效应。我们将建立一个低成本、高效的斑马鱼体内转基因系统来测试这些假定的功能性非编码序列。我们还将生成一个小鼠模型来测试绝缘体和消声器，这是目前标准小鼠转基因技术无法提供的。最后，我们将对ChIP芯片策略进行修改，通过将表位标签整合到各种类型的DNA结合蛋白中，允许它们在ChIP芯片实验中独立于针对每种被测试因子提出的特异性抗体的可用性。结合起来，这些策略将产生一个全面而强大的工具包，可用于生成几乎所有dna结合蛋白的全基因组顺式调控图谱，为鉴定其结合位点提供一个有效的系统，以及为验证ChIP芯片和计算实验所做的预测提供各种实验分析。

DESCRIPTION (provided by applicant): The initial phases of the ENCODE project resulted in the development and establishment of several high throughput strategies for the rapid identification of predicted functional elements in the human genome. Adequate systems to experimentally validate these predictions are currently lacking, especially methods that can test for elements that act as transcriptional silencers and boundary elements. We propose to generate an in vitro and in vivo platform to allow the efficient testing of predicted enhancer, silencers and insulators in the human genome. We will target a panel of 4 human cell lines as well as mouse embryonic stem cells with reporter vectors integrated in site-specific manner through homologous recombination. By using various vector designs we will develop cell lines that can be used for testing genomic sequences both for transcriptional activation properties as well as silencing and boundary properties, independent of variegation and multiple-copy integration effects. We will establish a low cost, efficient in vivo zebrafish transgenic system to test these putative functional noncoding sequences. We will also generate a mouse model to test insulators and silencers, currently not afforded by standard mouse transgenesis technologies. Finally, we will implement a modification on ChIP chip strategies, by integrating epitope tags to various classes of DNA binding proteins, allowing their use in ChIP chip experiments independent of the availability of specific antibodies raised against each factor being tested. Combined, these strategies will generate a comprehensive and powerful toolkit that can be used for the generation of genome-wide cis-regulatory maps of virtually all DNA-binding proteins, providing an efficient system for the identification of their binding sites as well as a variety of experimental assays for the validation of the predictions made by ChIP chip and computational experiments.