Novel technology for analysis of cardiac & BMP-specific gene expression profiling

心脏分析新技术

• 批准号: 8656805
• 负责人: TODD Andrew TOWNSEND
• 金额: $ 3.44万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2014-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8656805
• 关键词: Address; Atherosclerosis; Basic Science; Bioinformatics; Biotin; Candidate Disease Gene; Cardiac; Cardiovascular system; Cause of Death; Cell Differentiation process; Cell Lineage; Cell physiology; Cells; Cessation of life; Childhood; Clinical; Communities; Congenital Abnormality; Congenital Heart Defects; Data; Development; Elements; Embryo; Environment; Enzymes; Epitopes; Event; Fishes; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Genetic Translation; Goals; Heart; Heat-Shock Response; Human; Label; Life; Ligase; Live Birth; Messenger RNA; Methods; Molecular Profiling; Morphogenesis; Pathway interactions; Peptides; Polyribosomes; Process; Proteins; RNA; Research; Ribosomal Proteins; Ribosomes; Role; Sampling; Signal Pathway; Signal Transduction; Specificity; Staging; Streptavidin; System; Technology; Testing; Therapeutic; Tissues; Training; Transforming Growth Factors; Transgenic Organisms; Translating; Wound Healing; Zebrafish; base; bone morphogenic protein; cardiogenesis; cell growth; comparative; gene discovery; genetic manipulation; genome-wide; genome-wide analysis; heart cell; insight; mRNA Expression; member; new technology; novel; novel strategies; promoter; response; stillbirth; tool; transcriptome sequencing; tumor progression; zebrafish development

DESCRIPTION (provided by applicant): Congenital heart malformations occur at a rate of approximately 1% of human live births and 8% of stillbirths. While several critical components of cardiovascular development have been elucidated, the gene expression profiles and signaling mechanisms that govern these processes is largely undetermined. One of the challenges is purifying tissue and lineage-specific samples during specific developmental stages to assess subtle changes in mRNA expression or translation. To address this challenge, we generated a new technology that captures gene expression profiles in zebrafish by purifying polysomes from specific cell lineages (in this case cardiac and BMP-responding lineages) in order to identify the mRNAs that are being translated during specific timepoints in development. To accomplish this we have built a two-component system in which Biotin Ligase Recognition Peptide (BLRP) epitope is attached to accessible regions of two independent ribosomal proteins (Rpls), Rpl18a and Rpl23 and the second component drives expression of BirA in a lineage or cell-signaling responsive manner. BirA is an enzyme that covalently adds biotin to the BLRP-tagged construct. Using stable transgenic zebrafish lines, the goal is to drive BLRP-Rpl ubiquitously, and to drive BirA in a cell-lineage restricted manner with tissue-specific transcriptional promoter elements (for example, specifically in cardiac cells, or temporally regulated by a heat shock promoter, or in response to specific cell signals). Thus, only defined cell lineages will have both the BRLP-tagged ribosomes and the BirA enzyme will have ribosomes that are tagged with biotin. Immunopurification (IP) of these biotin-labeled ribosomes will allow us to purify actively translated mRNAs. As proof of principle, we tested the hypotheses that we can use BirA to biotinylate specific Rpl's in fish and that we can use a heart specific (cmlc2) promoter-driven BirA expression to label and IP polysomes from the heart lineage. Our first proposed aim is to investigate the cardiac-specific expression profiles of IP'ed polysomes using RNA-Seq at critical early developmental timepoints. Further, it is known that members of the TGF¿ superfamily, such as Bone Morphogenic Proteins (BMPs), control distinct cellular processes such as cell growth and differentiation to regulate development. Therefore, our second aim is to capture the gene expression profile of cells that are actively signaling via the BMP pathway, and perform comparative analysis versus other cell-signaling pathways. This project will generate a novel approach to discover gene expression profiles and translational control in the whole heart and specific heart lineages. The training environment is superb, with expertise in cardiovascular research and bioinformatics and genome-wide analyses. The research aims will yield translational profiles that will identify candidate genes and novel pathways in heart development and cell-signaling dependent morphogenesis. These results will have significant impact in basic research, provide tools for the zebrafish and cardiovascular research communities, and suggest new pathways for potential targets of clinical therapeutics.

描述（由申请人提供）：先天性心脏畸形的发生率约为1%的人类活产和8%的死产。虽然已经阐明了心血管发育的几个关键组成部分，但控制这些过程的基因表达谱和信号传导机制在很大程度上尚未确定。挑战之一是在特定发育阶段纯化组织和谱系特异性样品，以评估mRNA表达或翻译的细微变化。为了应对这一挑战，我们开发了一种新技术，通过纯化来自特定细胞谱系（在这种情况下是心脏和BMP反应谱系）的多聚核糖体来捕获斑马鱼的基因表达谱，以鉴定在发育的特定时间点正在翻译的mRNA。为了实现这一点，我们建立了一个双组分系统，其中生物素连接酶识别肽（BLRP）表位连接到两个独立的核糖体蛋白（Rpl）Rpl 18 a和Rpl 23的可接近区域，第二组分以谱系或细胞信号传导响应方式驱动BirA的表达。BirA是将生物素共价添加到BLRP标记的构建体的酶。我们的目标是利用稳定的转基因斑马鱼细胞系，使BLRP-Rpl基因在斑马鱼中无处不在地表达，并利用组织特异性转录启动子以细胞系限制的方式表达BirA基因 在一些实施方案中，这些基因可以是热激元件（例如，特异性地在心脏细胞中，或由热激启动子暂时调节，或响应于特定的细胞信号）。因此，只有确定的细胞谱系将具有这两个 BRLP标记的核糖体和BirA酶将具有用生物素标记的核糖体。这些生物素标记的核糖体的免疫纯化（IP）将使我们能够纯化主动翻译的mRNA。作为原理的证明，我们测试了以下假设：我们可以使用BirA来生物素化鱼中的特异性Rpl，并且我们可以使用心脏特异性（cmlc 2）启动子驱动的BirA表达来标记和IP来自心脏谱系的多核糖体。我们的第一个目标是使用RNA-Seq在关键的早期发育时间点研究IP'ed艾德多核糖体的心脏特异性表达谱。此外，已知TGF β超家族的成员，如骨形态发生蛋白（BMP），控制不同的细胞过程，如细胞生长和分化，以调节发育。因此，我们的第二个目标是捕获通过BMP途径主动信号传导的细胞的基因表达谱，并与其他细胞信号传导途径进行比较分析。该项目将产生一种新的方法来发现整个心脏和特定心脏谱系中的基因表达谱和翻译控制。培训环境一流，拥有心血管研究和生物信息学以及全基因组分析方面的专业知识。研究目标将产生翻译谱，将确定候选基因和心脏发育和细胞信号传导依赖的形态发生的新途径。这些结果将对基础研究产生重大影响，为斑马鱼和心血管研究社区提供工具，并为临床治疗的潜在靶点提供新的途径。