LAMININ ALPHA 2 IN DEVELOPMENT

这项研究的长期目标是确定分子 基底膜对组织的重要性背后的机制 发展和功能。本申请集中在层粘连蛋白上， 具体地说是层粘连蛋白α2链。Alpha2链是 来自Engelbreth-Holm的经典层粘连蛋白的A或α1链。 丛集性(EHS)肿瘤，在成熟的纹状组织中表达最明显 肌肉和周围神经。层粘连蛋白α2链受 人类和动物的某些肌肉疾病。在患者身上减少。 伴福山先天性肌营养不良并失联的患者 与其他某些先天性肌营养不良症有关。两个等位基因小鼠 存在肌营养不良突变：Dy，具有严重的表型和 极大地减少了层粘连蛋白α2链，并具有较温和的表型和 截短层粘连蛋白α2链。Dy2J小鼠的RNA发生了突变 导致mRNA异常剪接和缺失的剪接共识序列 在α2链的结构域VI中。目标L：我们将使用dy和dy2j 突变体研究层粘连蛋白的结构和功能。组织 层粘连蛋白在突变小鼠中的定位将通过光来研究。 以及与其他基底膜蛋白相关的电子显微镜。 电子显微镜将被用来研究孤立的， 突变的层粘连蛋白。突变层粘连蛋白与自身及其他蛋白的相互作用 基底膜蛋白和与细胞的关系将在#年进行研究 聚合、亲和力和细胞黏附试验。目标2：我们将 生成层粘连蛋白α2链缺失突变小鼠以确定其效果 在发育过程中完全缺乏这种层粘连蛋白链。其中一部分是 层粘连蛋白α2链基因的编码序列将被 LacZ基因在胚胎干细胞中的同源重组。这个 将分析纯合子零突变小鼠的表型并 与dy和dy2J小鼠相比。杂合子小鼠将会是 分析了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α2链。目的3：自发性缺陷和实验性缺陷 层粘连蛋白α2链缺陷小鼠将被部分纠正 野生型α2链的表达。转基因小鼠过表达 将产生层粘连蛋白α2链。这些小鼠将被培育成甲型2 突变小鼠产生表型更受限制的小鼠。结果： 自发和实验突变的小鼠将产生更好的 层粘连蛋白在不同组织中的功能和活性的知识 并可能导致更好的理解、诊断和治疗 肌肉营养不良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.