Dissecting of the Tbx20 Regulatory Network

剖析 Tbx20 监管网络

基本信息

批准号：
7851314
负责人：
Marcelo A. Nobrega
金额：
$ 43.68万
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 2012-06-30
项目状态：
已结题

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识别仍然不完整，部分原因是很难识别心脏发生过程中基因区域化表达所必需的关键顺式作用序列。在这里，我们提出了一个综合系统，以识别TBX20调节网络的关键组成部分，该基因在心脏发育中起着重要作用。为了实现这一目标，我们建议利用体内斑马鱼和小鼠记者测定法的组合来识别TBX20基因座中的心脏增强剂。我们将进行TBX20基因座中增强子的饱和扫描，无论其进化保守模式如何，都会系统地测试序列，目的是识别在心脏病发生过程中协调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空间和时间结构域进行详细的表征，从而对TBX20调节网络的组成部分有广泛的了解。我们将测试这些增强子中每个增强子的必要性，以适当地表达TBX20。使用工程的细菌人工染色体（BAC），我们将删除每个增强子，以驱动心脏多个领域的表达。这种实验设计将使我们能够推断监管网络的各个组件的必要性，以适当的时间，空间和定量表达TBX20。总而言之，我们将使用体内测定中的斑马鱼和小鼠来表征CIS调控网络，该网络在心脏病发生过程中坐标TBX20表达，并评估网络的各个组件如何协调TBX20表达的动态和复杂模式。