NOVEL RNA APPROACH THAT PROMOTES HEART DEV

促进心脏发育的新 RNA 方法

• 批准号: 2723695
• 负责人: LARRY F LEMANSKI
• 金额: $ 22.32万
• 依托单位: TEXAS ENGINEERING EXPERIMENT STATION
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-03-15 至 2002-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2723695
• 关键词: RNA; Urodela; cell differentiation; endoderm; gene deletion mutation; gene expression; genetic promoter element; genetic regulation; genetic transcription; genetically modified animals; in situ hybridization; invertebrate embryology; molecular cloning; myocardium; myofibrils; myogenesis; point mutation; polymerase chain reaction; tropomyosin

DESCRIPTION: (Adapted from Investigator's Abstract). The long term goal of the laboratory is to elucidate the cellular, molecular and genetic mechanisms that regulate myocardial cell differentiation and myofibrillogenesis in the developing heart. Ambystoma mixicanum is an intriguing model for studying heart development because it carries a mutation in gene c thought tot exert its effect via abnormal inductive processes from the anterior endoderm. The hearts of double recessive (c/c) mutant embryos do not beat, as they are deficient in tropomyosin and do not contain organized myofibrils. However, the defect can be corrected by culturing the mutant hearts in the presence of normal anterior endoderm tissue, medium conditioned by the anterior endoderm, or total RNA isolated from endoderm or the conditioned medium, each of which promotes myofibrillogenesis in the mutant hearts. A single clone (#4) has been identified from a cDNA library constructed from total conditioned medium RNA which can act as a template for the bioactive RNA capable of correcting the heart defect in organ culture as well as in vivo. The bioactive RNA can bind with a protein present in the embryonic axolotl heart and the bioactive RNA enters the cells of mutant hearts to effect rescue. It is hypothesized that the bioactive RNA is a regulatory molecules which up regulates tropomyosin synthesis in the mutant hearts for promoting myofibrillogenesis either directly or in complex with its binding protein. There are four Specific Aims to test this hypothesis: 1) The expression of the bioactive RNA will be examined in normal and mutant hearts at various stages of development using RT-PCR and in situ hybridization. Ectopic expression of sense and antisense RNA will be tested by creating transgenic axolotls; 2) In vitro mutagenesis of the clone #4 RNA will be performed to elucidate the mechanism of its rescuing activity as well as its binding ability to the newly-discovered binding protein; 3) Study up regulation of tropomyosin in the mutant hearts corrected by the cone #4 RNA; 4) The full length cDNA and the RNA and its mechanism by which its expression is regulated. It is anticipated that these studies will provide significant new information the mechanism of in vivo inductive interactions responsible for normal cardiac myocyte differentiation at the molecular level.

描述：（根据研究者的摘要进行了改编）。长期目标的 该实验室将阐明细胞，分子和遗传 调节心肌细胞分化和 发育中的心脏中的肌原纤维化。 Ambystoma Bimicicanum是 研究心脏发育的有趣模型，因为它带有 基因C思想中的突变通过异常诱导作用 从前内胚层进行过程。双隐性的心（C/C） 突变胚胎不击败，因为它们缺乏肌动蛋白并做 不包含有组织的肌原纤维。但是，缺陷可以通过 在正常内胚层存在下培养突变心 组织，由前内胚层调节或分离的总RNA 来自内胚层或条件培养基，每种培养基促进 突变心中的肌原纤维化。一个克隆（＃4）已经 从总条件培养基构建的cDNA库中确定 RNA可以充当能够生物活性RNA的模板 纠正器官培养和体内的心脏缺陷。这 生物活性RNA可以与存在于胚胎轴突中的蛋白质结合 心脏和生物活性RNA进入突变心的细胞以实现 救援。假设生物活性RNA是一种调节 调节突变心中tropomyosin合成的分子 用于促进肌原纤维化直接或与其复杂 结合蛋白。有四个特定的目的来检验这一假设：1） 生物活性RNA的表达将在正常和突变体中检查 使用RT-PCR和原位开发的各个阶段的心脏 杂交。感官和反义RNA的异位表达将是 通过创建转基因Axolotls测试； 2）体外诱变的 克隆＃4 RNA将进行阐明其营救机制 活动以及与新发现的结合的结合能力 蛋白质; 3）研究突变心脏中肌球蛋白的调节 通过锥体＃4 RNA校正； 4）全长cDNA和RNA及其 通过调节其表达的机制。预计 这些研究将提供重要的新信息 负责正常心肌细胞的体内诱导相互作用 分子水平的分化。