Functional Analysis of Collagen IV by Gene Targeting

通过基因打靶对 IV 型胶原蛋白进行功能分析

基本信息

批准号：
09044308
负责人：
NINOMIYA Yoshifumi
金额：
$ 3.26万
依托单位：
Okayama University
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09044308/
关键词：
collagen IV basement membrane knockout mouse Alport syndrome Leiomyomatosis smooth muscle cell gene targeting molecular form 分子型コラーゲン遺伝子 COL4A6 平滑筋腫ノックアウト

项目摘要

Col4a6 null mice were created by introducing NeoィイD1RィエD1 gene into exon II of col4a6 gene. We analyzed if the gene is transcribed and translated into α6(IV) polypeptide by in site hybridization, Northern-blot hybridization, and immunohistochemical staining using α chain-specific monoclonal antibodies. The results demonstrated that the α6(IV) polypeptide was not stained at all in any tissues or organs in col4a6 null mice. We also analyzed if the mice have some phenotypes, but we have not detected ant differences between wild type and col4a6 null mice so far.We predicted the presence of the three molecular forms of type IV collagen ; [α1(IV)]2α2(IV), α3(IV)α4(IV)α5(IV), and [α5(IV)]2α6α(IV) by use of double staining of the monoclonal antibodies specific for α(IV) chains. The [α1(IV)]2α2(IV) form is present in all basement membranes, whereas the α3(IV)α4(IV)α5(IV) form is localized in glomeruler and alveolar basement membranes and the [α5(IV)]2α6(IV) form is in the Bowman's capsules of the kidney and dermal basement membrane regions. Heterogeneous distribution of the three molecular forms indicated that they may have specific roles in different basement membranes. To describe the biological roles of the molecules, we should think of how the three molecules are incorporated into the supramolecular aggregates of each basement membrane.We identified and characterized the breakpoint sequences of the deletion of DNA from a patient of diffuse leiomyomatosis associated with Alport syndrome. The results demonstrated that a deletion eliminates the first coding exon of COL4A5 and the first two of COL4A6. They also showed that the breakpoints share the same sequence, which is in turn closely homologous to the consensuses of topoisomerases I and II. Additional DNA evidence suggested that the male patient is a somatic for the mutation. This study is greatly relevant to the understanding of DL pathogenesis and its etiology.

通过将 NeoiiD1RieD1 基因引入 col4a6 基因的外显子 II 来创建 Col4a6 缺失小鼠，我们通过原位杂交、Northern 印迹杂交和使用 α 链特异性单克隆抗体的免疫组织化学染色分析该基因是否转录并翻译为 α6(IV) 多肽。结果表明，在 col4a6 缺失小鼠的任何组织或器官中，α6(IV) 多肽均未染色。小鼠具有一些表型，但迄今为止我们尚未检测到野生型和 col4a6 缺失小鼠之间的差异。我们预测存在 IV 型胶原的三种分子形式；[α1(IV)]2α2(IV)、α3( IV)α4(IV)α5(IV) 和 [α5(IV)]2α6α(IV)，通过使用对 α(IV) 链特异的单克隆抗体进行双重染色。 [α1(IV)]2α2(IV) 形式存在于所有基底膜中，而 α3(IV)α4(IV)α5(IV) 形式位于肾小球和肺泡基底膜中，[α5(IV)]2α6 形式位于肾小球和肺泡基底膜中。 (IV)形式在肾和真皮基底膜区域的鲍曼囊中的三种分子形式的异质分布表明它们可能在不同的基底膜中具有特定的作用。考虑到这些分子的生物学作用，我们应该考虑这三个分子是如何整合到每个基底膜的超分子聚集体中的。我们鉴定并表征了与阿尔波特综合征相关的弥漫性平滑肌瘤病患者的 DNA 缺失断点序列。删除消除了 COL4A5 的第一个编码外显子和 COL4A6 的前两个编码外显子。他们还表明，断点共享相同的序列，这反过来又与 COL4A6 的共有序列密切同源。拓扑异构酶 I 和 II。额外的 DNA 证据表明，该男性患者是体细胞突变。这项研究与了解 DL 发病机制及其病因学密切相关。