Dissecting of the Tbx20 Regulatory Network

剖析 Tbx20 监管网络

• 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
• 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

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.

协调心脏发育的遗传途径的层次结构的认识仍然是不完整的，部分原因是难以确定关键的顺式作用序列，所需的区域化表达的基因在心脏发生。在这里，我们提出了一个综合系统，以确定TBX20，在心脏发育中发挥重要作用的基因的调控网络的关键组成部分。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子。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，而不管它们的进化保守模式如何，目的是确定在心脏发生过程中协调TBX20表达的关键增强子。我们将在胚胎发育过程中对这些心脏增强子的空间和时间域进行详细的表征，从而对TBX20调控网络的组成部分有广泛的了解。我们将测试这些增强子中的每一个对于TBX20的适当表达的必要性。使用工程化的细菌人工染色体（BAC），我们将删除驱动心脏多个结构域表达的每个增强子。这个实验设计将使我们能够推断出TBX20的适当的时间，空间和定量表达的调控网络的各个组件的必要性。 总之，我们将使用斑马鱼和小鼠体内试验来表征心脏发生过程中协调TBX20表达的顺式调节网络，并评估网络的各个组成部分如何协调TBX20表达的动态和复杂模式。