STUDIES OF A NOVEL RNA APPROACH THAT PROMOTES HEART DEV

促进心脏发育的新型 RNA 方法的研究

• 批准号: 6457618
• 负责人: LARRY F LEMANSKI
• 金额: $ 21.4万
• 依托单位: FLORIDA ATLANTIC UNIVERSITY
• 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 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6457618
• 关键词: RNA; Urodela; cardiac myocytes; 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：（改编自研究者的摘要）。长期目标是 该实验室的目的是阐明细胞、分子和遗传 调节心肌细胞分化的机制 发育中的心脏中的肌原纤维发生。 Ambystoma mixicanum 是一种 研究心脏发育的有趣模型，因为它带有 基因c突变被认为不是通过异常诱导发挥其作用 来自前内胚层的突起。双隐性心 (c/c) 突变胚胎不会跳动，因为它们缺乏原肌球蛋白并且 不含有组织的肌原纤维。然而，该缺陷可以通过以下方式纠正： 在正常前内胚层存在的情况下培养突变心脏 组织、前内胚层条件培养基或分离的总 RNA 来自内胚层或条件培养基，两者均促进 突变心脏中的肌原纤维发生。单个克隆（#4）已被 从总条件培养基构建的 cDNA 文库中鉴定 RNA 可以作为生物活性 RNA 的模板 在器官培养和体内纠正心脏缺陷。这 生物活性RNA可以与胚胎蝾螈中存在的蛋白质结合 心脏和生物活性RNA进入突变心脏的细胞以发挥作用 救援。据推测，生物活性RNA是一种调节性RNA。 上调突变心脏中原肌球蛋白合成的分子 直接或与其联合促进肌原纤维生成 结合蛋白。有四个具体目标来检验这一假设：1) 将在正常和突变体中检查生物活性 RNA 的表达 使用 RT-PCR 和原位观察不同发育阶段的心脏 杂交。正义和反义RNA的异位表达将是 通过创建转基因蝾螈进行测试； 2) 体外诱变 将进行克隆 #4 RNA 以阐明其拯救机制 活性及其与新发现的结合的结合能力 蛋白质; 3) 研究突变心脏中原肌球蛋白的上调 由锥#4 RNA 校正； 4) 全长cDNA和RNA及其 其表达受到调节的机制。预计 这些研究将为这一机制提供重要的新信息 负责正常心肌细胞的体内诱导相互作用 分子水平上的分化。