DIFFERENTIALLY EXPRESSED GENES IN CARDIAC MORPHOGENESIS

心脏形态发生中的差异表达基因

• 批准号: 6565115
• 负责人: Jim Jung-Ching Lin
• 金额: $ 44.31万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-01-01 至 2002-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6565115
• 关键词: DNA binding protein; RNase protection assay; antisense nucleic acid; cell differentiation; congenital heart septum defect; developmental genetics; embryo /fetus tissue /cell culture; gene expression; gene targeting; genetic screening; genetically modified animals; heart; histogenesis; immunofluorescence technique; in situ hybridization; laboratory mouse; laboratory rabbit; linkage mapping; mammalian embryology; protein localization; protein sequence; transcription factor

(Adapted from the Applicant's Abstract) Functional mammalian hearts require successful septation and valvulogenesis to form four chambers and a fully differentiated myocardium. The long-term goal is to understand underlying mechanisms for septation, valvulogenesis and differentiation. The investigators previously cloned several novel, differentially-expressed genes (DEGs) including Xin. Initial studies on Xin reveal that it may play important roles in cardiac looping. Incomplete looping has been implicated in septal misalignment/defect in several mouse models. Thus, mouse homologs of these genes together with additional genes to be cloned in Project 2 of this SCOR will be further characterized to understand their roles in cardiac morphogenesis and possibly to reveal their relevance to the etiology of atrial septal, AV canal and ventricular septal defects. Aim 1 is to determine temporal and spatial expression patterns of DEGs. Large-scale, whole-mount in situ screening on DEGs, initially cloned by Project 2 from developing rat hearts, will be carried out. These DEG clones will be prioritized and selected clones will be further characterized. For example, detail in situ hybridization and RNASE protection assay will be used to determine how early the Xin expresses, when its expression reaches maximum and where it is. Simultaneously, full-length mouse clones will be isolated and used for protein and antibody productions. Immunofluorescence will be performed with specific antibody. Aim 2 is to examine the properties of DEG proteins. The predicted protein sequence will be searched for known motifs for which experiments will be designed to test. For example, nuclear localization signal found in Xin has been shown to be functional. Once antibody is available, localization of Xin can be further determined by immunofluorescence. Studies on DNA binding and transcriptional activities of Xin are also proposed. Relationship of Xin to other genes in the controlling pathway for looping will be studied in Nkx2.5-, MEF2C-, or dHAND-knockout mice. Aim 3 is to perform assays to assess roles of DEGs in cardiac morphogenesis, including antisense studies, epithelial-mesenchymal cell transformation, and knockout and transgenic mice.

（改编自申请人的摘要）功能性哺乳动物心脏需要成功的分离和瓣膜发生，以形成四个腔室和完全分化的心肌。 长期目标是了解分隔，阀发生和分化的基本机制。 研究人员先前以包括XIN在内的几个新型，差异表达的基因（DEG）克隆。 对XIN的初步研究表明，它可能在心脏循环中起重要作用。在几种鼠标模型中，不完整的循环已与间隔未对准/缺陷有关。 因此，这些基因的小鼠同源物以及在该SCOR的项目2中克隆的其他基因将进一步特征，以了解其在心脏形态发生中的作用，并可能揭示其与房屋中隔膜，AV管和心室缺陷的病因的相关性。 目的1是确定DEG的时间和空间表达模式。 将在DEG上进行大规模的全部原位筛选，最初由Deverence Rat Hearts的项目2克隆。 这些DEG克隆将优先考虑，并将进一步表征选定的克隆。 例如，将使用细节原位杂交和RNase保护分析来确定XIN表达的早期表达方式，何时达到最大值和位置。同时，将分离出全长小鼠克隆并用于蛋白质和抗体生产。免疫荧光将使用特定抗体进行。 目标2是检查DEG蛋白的特性。 将搜索预测的蛋白质序列，以搜索已知的实验序列的已知基序。 例如，XIN中发现的核定位信号已被证明是功能性的。 一旦获得抗体，可以通过免疫荧光进一步确定XIN的定位。 还提出了XIN的DNA结合和转录活性的研究。 XIN与循环控制途径中其他基因的关系将在NKX2.5-，MEF2C-或DHOND-KNOCKOUT小鼠中进行研究。 AIM 3是进行测定，以评估DEG在心脏形态发生中的作用，包括反义研究，上皮 - 间质细胞转化以及敲除和转基因小鼠。