Dissecting of the Tbx20 Regulatory Network

剖析 Tbx20 监管网络

基本信息

批准号：
7373446
负责人：
Marcelo A. Nobrega
金额：
$ 40.69万
依托单位：
UNIVERSITY OF CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-07-01 至 2011-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7373446
关键词：
Address Bacterial Artificial Chromosomes Bioinformatics Biological Assay Biology Cardiac Cardiac Myocytes Cause of Death Child Cis-Acting Sequence Classification Code Communities Complex Congenital Abnormality DNA Data Development Distant Elements Embryo Embryonic Development Engineering Enhancers Exons Experimental Designs Fishes Gene Expression Genes Genetic Genetic Engineering Genetic Transcription Goals Growth Heart Human In Vitro Inborn Genetic Diseases Individual Junk DNA Libraries Maps Molecular Morphogenesis Mus Mutation Pathway interactions Pattern Performance Plasmids Play Process Property Proteins Reagent Regulator Genes Regulatory Element Reporter Role Scanning Shuttle Vectors Stereotyping Structure System Techniques Testing Transcriptional Regulation Transgenic Mice Transgenic Organisms Zebrafish base cardiogenesis congenital heart disorder design human disease in vivo novel research study transcription factor

项目摘要

The recognition of the hierarchies of genetic pathways coordinating heart development remains incomplete, in part because of the difficulties to identify the critical cis-acting sequences that are required for the regionalized expression of genes during cardiogenesis. Here we propose an integrative system to identify the critical components of the regulatory network of TBX20, a gene that plays important roles in heart development. To achieve this, we propose to utilize a combination of an in vivo zebrafish and mouse reporter assay to identify heart enhancers in the TBX20 locus. We will carry out a saturation scan for enhancers in the TBX20 locus, systematically testing sequences regardless of their pattern of evolutionary conservation, with the goal of identifying the critical enhancers that coordinate TBX20 expression during cardiogenesis. We will carry out a detailed characterization of the spatial and temporal domains of these heart enhancers during embryogenesis, leading to a broad understanding of the components of the TBX20 regulatory network. We will test the necessity of each of these enhancers for the proper expression of TBX20. Using engineered Bacterial Artificial Chromosomes (BACs), we will delete each enhancer that drives expression in multiple domains of the heart. This experimental design will allow us to infer the necessity of individual components of the regulatory network for the proper temporal, spatial and quantitative expression of TBX20. In summary, we will use zebrafish and mouse in vivo assays to characterize the cis-regulatory network that coordinates TBX20 expression during cardiogenesis, and evaluate how the individual components of the network orchestrate the dynamic and complex pattern of TBX20 expression.

对协调心脏发育的遗传途径层次结构的认识仍然不完整，部分原因是难以识别心脏发生过程中基因区域化表达所需的关键顺式作用序列。在这里，我们提出了一个综合系统来识别 TBX20 调控网络的关键组成部分，TBX20 是一种在心脏发育中发挥重要作用的基因。为了实现这一目标，我们建议利用体内斑马鱼和小鼠报告基因检测的组合来识别 TBX20 基因座中的心脏增强剂。我们将对 TBX20 基因座中的增强子进行饱和扫描，系统地测试序列，无论其进化保守模式如何，目的是确定在心脏发生过程中协调 TBX20 表达的关键增强子。我们将对胚胎发生过程中这些心脏增强剂的时空域进行详细表征，从而对 TBX20 调控网络的组成部分有更广泛的了解。我们将测试每个增强子对于 TBX20 正确表达的必要性。使用工程细菌人工染色体（BAC），我们将删除驱动心脏多个区域表达的每个增强子。该实验设计将使我们能够推断调控网络各个组成部分对于 TBX20 正确的时间、空间和定量表达的必要性。总之，我们将使用斑马鱼和小鼠体内测定来表征在心脏发生过程中协调 TBX20 表达的顺式调控网络，并评估该网络的各个组件如何协调 TBX20 表达的动态和复杂模式。