Additional Tool Development or Data Generation

额外的工具开发或数据生成

• 批准号: 9020717
• 负责人: David M Gilbert
• 金额: $ 32.77万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-28 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9020717
• 关键词: Address; Benchmarking; Binding; Biological; Cell Line; Cells; Chromosomes; Collection; Communities; Custom; DNA; DNA Sequence; DNA biosynthesis; Data; Data Set; Development; Diploid Cells; Dissection; Elements; Foundations; Generations; Genome; Genome Mappings; Genomic Segment; Genomics; Haploidy; Human; Imagery; Individual; Life; Maps; Methods; Microscopy; Nuclear; Nuclear Lamina; Paint; Phototoxicity; Proteins; Protocols documentation; Reagent; Series; Signal Transduction; Staining method; Stains; Stretching; Tandem Repeat Sequences; Technology; Validation; abstracting; cell fixing; cellular imaging; design; functional genomics; genome analysis; genome-wide; improved; in vivo; insight; novel; tool; tool development

PROJECT SUMMARY / ABSTRACT – ADDITIONAL TOOL DEVELOPMENT OR DATA GENERATION In this Additional Tool Development or Data Generation (ATDG) module we will focus on the development of several advanced technologies that will further boost the analysis of genome compartmentalization. First, we will address the urgent need to manipulate large genomic regions in order to gain insights into the mechanisms and functions of genome compartmentalization. It is likely that the targeting of genomic loci to specific compartments is encoded in multiple sequence elements dispersed over long stretches of DNA. Identification of these targeting signals will therefore require extensive manipulation of long genomic regions. We will develop a pipeline for the custom design and automated assembly of ~100kb DNA sequences, which will enable us to freely edit, delete, or add multiple sequence elements. In addition, we will establish protocols and cell lines for the easy integration of such large DNA regions into the genome. Combined, these tools will allow for the systematic dissection of the mechanisms responsible for genome compartmentalization. Second, we will develop an extensive set of reagents for easy visualization and live-cell tracking of individual genomic loci. Using our automated DNA synthesis pipeline, we will generate a large collection of TALE probes that each bind to a single-locus tandem repeat. This will enable the in vivo microscopy of hundreds of specific genomic loci. For substantially higher sensitivity, we will develop improved genome visualization using split-GFP TALEs. Additionally, we will develop staining of fixed cells using pools of purified TALE proteins. Third, we will address the need to construct maps of genome – compartment associations in single cells; such maps are crucial for our understanding of cell-to-cell variability of genome organization. We have recently established a modified DamID protocol that can map nuclear lamina interactions genome-wide in single cells. As this protocol has so far only been used in haploid human cells, we will adapt it for use in diploid cells, using SNPs to discriminate homologous chromosomes. We will then apply this protocol to map genome interactions with several nuclear compartments in series of single cells. These three technologies will greatly enhance our abilities to manipulate, visualize and map regions of the genome that associate with specific nuclear compartments. They will not only pave the way for advanced studies in the Biological Validation Development module and beyond, but also yield novel and unique protocols, reagents and data sets for the scientific community.

项目总结/摘要-其他工具开发或数据生成 在此附加工具开发或数据生成（ATDG）模块中，我们将重点关注 开发几种先进技术，进一步推动基因组分析 划分 首先，我们将解决操纵大基因组区域的迫切需要，以便深入了解 基因组区室化的机制和功能。很可能，基因组位点的靶向， 特定的区室被编码在分散在长段DNA上的多个序列元件中。 因此，这些靶向信号的鉴定将需要对长基因组区域进行广泛的操作。 我们将开发一个定制设计和自动组装~100kb DNA序列的管道， 将使我们能够自由地编辑、删除或添加多个序列元素。另外，我们会制定 和细胞系，以便将这样大的DNA区域容易地整合到基因组中。这些工具将 允许系统解剖负责基因组区室化的机制。 其次，我们将开发一套广泛的试剂，以便于可视化和活细胞跟踪。 单个基因座。使用我们的自动化DNA合成管道，我们将生成大量的 TALE探针，其各自结合至单基因座串联重复。这将使体内显微镜的 数百个特定的基因位点为了获得更高的灵敏度，我们将开发改进的基因组 使用split-GFP TALE进行可视化。此外，我们将使用纯化的 TALE蛋白。 第三，我们将解决需要构建基因组分区协会在单一的 这些图谱对于我们理解基因组组织的细胞间变异性至关重要。我们有 最近建立了一个修改的DamID协议，可以映射核纤层相互作用的基因组范围内， 单细胞由于该方案迄今为止仅用于单倍体人类细胞，我们将使其适用于二倍体细胞。 细胞，使用SNP来区分同源染色体。然后我们将应用该协议来绘制基因组图谱 与一系列单细胞中的几个核隔室的相互作用。 这三项技术将大大提高我们操纵、可视化和绘制区域地图的能力。 与特定核区室相关的基因组。他们不仅会为先进的 生物学验证开发模块及其他模块中的研究，而且还产生了新颖独特的 为科学界提供的方案、试剂和数据集。