CARDIAC DEVELOPMENT DURING SITUS INVERSUS EMBRYOGENESIS

逆位胚胎发生期间的心脏发育

• 批准号: 6593872
• 负责人: Paul A. Overbeek
• 金额: $ 17.52万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6593872
• 关键词: artificial chromosomes; cellular polarity; congenital heart disorder; developmental genetics; embryogenesis; gene expression; gene targeting; genetic mapping; genetically modified animals; heart; laboratory mouse; mammalian embryology; myogenesis; polymerase chain reaction; restriction fragment length polymorphism; southern blotting

Critical events in early development include the initiation of looping in the embryonic heart tube, and the concomitant establishment of left-right asymmetry. Although the molecular events that determine the directions of looping remain unidentified, a number of genes have recently been identified that exhibit left-right asymmetric expression prior to the onset of heart looping. However, these asymmetrically expressed genes do not appear to be responsible for the initial specification of left-right asymmetry. Developmental studies of transgenic mice that carry the inv (inversion of embryonic rotation) insertional mutation suggest that the inv gene is essential for the earliest stages of left-right axis specification. Homozygous inv embryos exhibit a consistent reversal of morphological and molecular left-right asymmetry. In order to begin to characterize the inv locus, the transgenic integration site was cloned and characterized. Mapping studies revealed that integration of the transgene was accompanied by a significant genomic deletion and an intrachromosomal duplication on mouse chromosome 4. Single-copy genomic sequences flanking the integration site was used to identify a YAC clone (GO571) that spanned the deleted region. This YAC was purified and used for microinjection to generate transgenic mice. Six founder mice that had integrated both arms of the YAC were identified. Breeding studies revealed that the G0571 YAC can cure the inv mutant phenotype. Therefore this YAC contains the coding and regulatory elements of the inv gene. In order to further characterize this gene and its role in establishing left-right polarity, the following specific aims are proposed: 1. To identify the coding sequences for the inv gene (by exon trapping and cDNA library screening). 2. To determine the embryonic patterns of expression of the candidate inv mRNA and protein. 3. To characterize changes in expression of asymmetrically expressed genes (Nodal, lefty, Snail, flectin and dHAND) in YAC-cured embryos, in chimeric embryos, and in double mutant (iv/iv;inv/inv) embryos. 4. To construct an inv minigene and to use the minigenes to identify and characterize the functional domains in the protein. These experiments should provide molecular details about the earliest steps in left-right axis specification and the control of cardiac morphogenesis.

早期发展的关键事件包括启动循环 胚胎心管，以及伴随的左右建立 不对称。尽管决定了决定方向的分子事件 循环保持身份不明，最近有许多基因 鉴定出在左右不对称表达之前 心脏循环的发作。但是，这些不对称表达的基因DO 似乎不负责左右的初始规范 不对称。携带INV的转基因小鼠的发展研究 （胚胎旋转的反转）插入突变表明 INV基因对于左右轴的最早阶段至关重要 规格。纯合INV胚胎表现出一致的逆转 形态学和分子左右不对称。为了开始 特征是Inv轨迹，将转基因整合位点克隆，并 特征。映射研究表明，转基因的整合 伴随着明显的基因组缺失和一个内骨体内体 小鼠染色体4的复制。单拷贝基因组序列两侧 集成站点用于识别跨越跨度的YAC克隆（GO571） 删除的区域。该YAC被纯化，用于对 产生转基因小鼠。六只已经整合了双臂的创始人老鼠 确定了YAC的。育种研究表明，G0571 YAC 可以治愈INV突变表型。因此，此YAC包含编码 和INV基因的调节元素。为了进一步表征 该基因及其在建立左右极性中的作用，以下 提出了具体目的：1。确定编码序列 INV基因（通过外显子捕获和cDNA库筛选）。 2。确定 候选INV mRNA和 蛋白质。 3。表征不对称表达的变化 YAC固化的表达基因（淋巴结，左撇子，蜗牛，触发蛋白和DHAND） 胚胎，在嵌合胚和双突变体（IV/IV; Inv/Inv）中 胚胎。 4。要构建一个Inv Minigene并使用微型元素 识别并表征蛋白质中的功能域。这些 实验应提供有关最早步骤的分子细节 左右轴规范和心脏形态发生的控制。