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特定细胞类型中体内顺式调节元件的功能活性。Powered by 下一代测序，一系列高通量方法可以生成数据，使研究人员能够预测基因组中潜在顺式调控元件的位置。总的来说，这些方法已经产生了数十万个预测。一个主要的问题是，没有相应的技术来验证这些预测在体内的顺式调节活性。为了解决这个问题，我们建议开发一个大规模平行的报告基因检测，以测试活性的顺式调控元件和它们的等位基因变体在体内，在特定的细胞类型。我们的计划是开发方法来创建大库的顺式调控元件在慢病毒为基础的报告，并开发方法来测定这些库的活性，从特定群体的细胞在体内。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，但该方法将推广到来自不同生物体不同组织的大范围细胞类型。成功完成 我们的目标是产生一种高通量方法，用于在特定细胞类型中测试体内顺式调节多态性的作用。由于很大一部分致病变异被认为存在于非编码DNA中，因此测试等位基因变异影响的可扩展方法 在顺式调节元件中的应用将对该领域产生巨大影响。

 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.