MASSIVELY PARALLEL CHARACTERIZATION OF CIS-REGULATORY ELEMENTS IN THE BRAIN

 描述(由申请人提供)：我们建议开发一种高通量方法来测试体内特定细胞类型中顺式调节元件的功能活性。由 下一代测序，一系列高通量方法可以产生数据，使研究人员能够预测潜在的顺式调控元件在基因组中的位置。总而言之，这些方法产生了数十万个预测。一个主要问题是，目前还没有相应的技术来验证这些预测在体内的顺式调控活性。为了解决这个问题，我们建议建立一种大规模平行的报告基因分析来测试体内特定细胞类型中顺式调节元件及其等位基因变体的活性。我们的计划是开发方法在基于慢病毒的记者中创建大型顺式调控元件文库，并开发方法从体内特定的细胞群体中分析这些文库的活性。我们建议首先开发这项技术来研究大脑特定细胞类型的顺式调节，但这种方法将推广到不同生物体中不同组织的大范围细胞类型。成功完成 我们的目标是建立一种高通量的方法来测试体内特定细胞类型中顺式调控多态的影响。由于很大一部分致病变异被认为存在于非编码的DNA中，这是一种可扩展的方法来测试等位基因变异的影响 在顺势监管因素方面，将对该领域产生很大影响。

 DESCRIPTION (provided by applicant): We propose to develop a high-throughput method to test the functional activity of cis-regulatory elements in vivo, in specific cell types. Powered by Next Generation Sequencing, a range of high-throughput methods can generate data that allow investigators to predict the locations of potential cis-regulatory elements in the genome. Collectively these methods have generated hundreds of thousands of predictions. A major problem is that there are no corresponding technologies to validate the cis-regulatory activity of these predictions in vivo. To address this problem we propose to develop a massively parallel reporter gene assay to test the activity of cis-regulatory elements and their allelic variants in vivo, in specific cell types. Our plan is to develop methods to create large libraries of cis regulatory elements in lentiviral-based reporters, and to develop methods to assay the activity of these libraries from specific populations of cells in vivo. We propose to develop this technology first to study cis-regulation in specific cell types of the brain, but the approach will generalizeto a large range of cell types from different tissues in different organisms. Successful completion of our aims will result in a high-throughput method for testing the effects of cis-regulatory polymorphism in vivo, in specific cell types. Since a large fraction of disease causing variants are thought to reside in non-coding DNA, a scalable method to test the effects of allelic variation in cis-regulatory elements would have a large impact on the field.