LAMININ ALPHA 2 IN DEVELOPMENT

这项研究的长期目标是确定 观察到的基底膜对组织的重要性背后的机制 发展和功能。本申请集中于层粘连蛋白， 特别是层粘连蛋白α 2链。α 2链是 来自Engelbreth-Holm的经典层粘连蛋白的A或α 1链， Swarm（EHS）肿瘤，在成熟的横纹肌中表达最显著。 肌肉和周围神经。层粘连蛋白α 2链受到 人类和动物的某些肌肉疾病。在患者中减少 与福山先天性肌营养不良症和失踪的病人 与其他先天性肌肉萎缩症有关双等位基因小鼠 存在肌营养不良的突变体：dy，具有严重的表型， 层粘连蛋白α 2链大大减少，dy 2 J具有较温和的表型， 截短的层粘连蛋白α 2链。dy 2 J小鼠的RNA发生了突变 导致mRNA异常剪接和缺失剪接共有序列 在α 2链的结构域VI中。目标1：我们将使用dy和dy 2 j 突变体研究层粘连蛋白的结构和功能。组织 突变小鼠中层粘连蛋白的定位将通过光 和电子显微镜与其他基底膜蛋白的关系。 电子显微镜将用于研究分离的， 突变的层粘连蛋白突变的层粘连蛋白与自身和其他蛋白的相互作用 基底膜蛋白和细胞将在 聚合、亲和和细胞粘附测定。目标2：我们 产生层粘连蛋白α 2链无效突变小鼠以确定其作用 层粘连蛋白链的缺失。的一部分 层粘连蛋白α 2链基因的编码序列将被替换为 LacZ基因在胚胎干细胞中的同源重组。的 分析纯合无效突变小鼠的表型， 与dy和dy 2 J小鼠相比。杂合子小鼠将 分析β-半乳糖苷酶的表达，特别是在早期 胚胎发生，以获得有关正常表达的信息， 层粘连蛋白α 2链。目的3：自发性和实验性的缺陷 层粘连蛋白α 2-链缺陷小鼠将被部分纠正， 野生型α 2链的表达。转基因小鼠过度表达 将产生层粘连蛋白α 2链。这些老鼠将被培育成α 2 突变小鼠以产生具有更多限制性表型的小鼠。结果与 自发和实验突变的小鼠将产生更好的 了解层粘连蛋白在不同组织中的功能和活性 并可能导致更好的理解，诊断和治疗 肌肉萎缩症和其他神经肌肉疾病。

The long term goal of this research is to determine the molecular mechanisms behind the observed importance of basement membranes for tissue development and function. The present application is focused on laminin, specifically the laminin alpha2 chain. The alpha2 chain is a homologue of the A or alpha1 chain of the classical laminin from the Engelbreth-Holm- Swarm (EHS) tumor and is expressed most prominently in mature striated muscle and peripheral nerve. The laminin alpha2 chain is affected in certain muscle diseases in humans and animals. It is reduced in patients with the Fukuyama congenital muscular dystrophy and missing in patients with certain other congenital muscular dystrophies. Two allelic mouse mutants with muscular dystrophy exist: the dy, with severe phenotype and greatly reduced laminin alpha2 chain, and dy2J with milder phenotype and a truncated laminin alpha2 chain. The dy2J mouse has a mutation in an RNA splice consensus sequence causing abnormal splicing of mRNA and deletions in the domain VI of the alpha2 chain. Aim l: We will use the dy and dy2J mutants to study structure and function of laminin. The tissue localization of the laminin in the mutant mice will be studied by light and electron microscopy in relation to other basement membrane proteins. Electron microscopy will be used to study the structure of isolated, mutated laminin. The interaction of mutated laminin with itself and other basement membrane proteins and with cells will be studied in polymerization-, affinity-, and cell adhesion assays. Aim 2: We will generate a laminin alpha2 chain null mutant mouse to determine the effect of complete absence of this laminin chain on development. A portion of the coding sequence of the laminin alpha2 chain gene will be replaced with the LacZ gene by homologous recombination in embryonic stem cells. The phenotype of the homozygous null mutant mouse will be analyzed and compared to that of the dy and dy2J mice. Heterozygous mice will be analyzed for expression of beta-galactosidase, particularly in early embryogenesis, to obtain information on the normal expression of the laminin alpha2 chain. Aim 3: The defect in spontaneous and experimental laminin alpha2-chain deficient mice will be partially corrected by expression of wild type alpha2 chain. Transgenic mice overexpressing laminin alpha2 chain will be generated. These mice will be bred to alpha2 mutant mice to produce mice with a more restricted phenotype. Results with spontaneous and experimentally mutated mice will result in better knowledge of the function and activities of laminin in different tissues and may lead to improved understanding, diagnosis, and treatment of muscular dystrophies and other neuromuscular diseases.