Biological function of basement membrane

基底膜的生物学功能

• 批准号: 07044268
• 负责人: NINOMIYA Yoshifumi
• 金额: $ 2.56万
• 依托单位: Okayama University Medical School
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for international Scientific Research
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07044268/
• 关键词: genomic library; monoclonal antibodies; homologous recombination; collagen genes; phynotypic change; knockout mouse; 遺伝子ライブラリ; cDNAライブラリー; IV型コラーゲン

1) We have cloned many overlapping cDNAs coding for the mouse alpha6 (IV) collagen chain and determined primary structure of the alpha6 (IV) chain polypeptide.2) We raised monoclonal antibodies against amino acid sequences from various parts of the mouse alpha6 (IV) collagen polypeptide chain and used them for the tissue distribution. The results indicated that the tissue distribution for the chain was similar to that of the human alpha6 (IV) collagen chain. Interestingly, the distribution of the paired genes ; col4a1/col4a2, col4a3/col4a4, and col4a5/col4a6 is quite synchronous except for the glomerular basement membranes.3) We have some indication that shows alternative splicing in exon 31. Currently, we are characterizing this atypical splicing event in terms of tissue specific expression.4) In order to create a knockout mice for col4a6, we made several constructs including the one where the exon 2 was taken out and the foreign gene, Neo^R gene was inserted instead. The construct was transfected into Stem cells by homologous recombination. Currently, we are characterizing knockout mice to detect any phenotypic changes in heterozygotes and homozygotes.

1)我们克隆了许多编码小鼠α 6（IV）胶原蛋白链的重叠cDNA，并确定了α 6（IV）链多肽的一级结构。2）我们提出了针对小鼠α 6（IV）胶原蛋白多肽链各个部分的氨基酸序列的单克隆抗体，并将它们用于组织分布。结果表明，该链的组织分布与人α 6（IV）胶原蛋白链的组织分布相似。有趣的是，配对基因col 4a 1/col 4a 2，col 4a 3/col 4a 4和col 4a 5/col 4a 6的分布是相当同步的，除了肾小球基底膜。目前，我们正在根据组织特异性表达来表征这种非典型剪接事件。4）为了创建col 4a 6基因敲除小鼠，我们制作了几种构建体，包括其中外显子2被取出并插入外源基因Neo^R基因的构建体。通过同源重组将构建体转染到干细胞中。目前，我们正在研究基因敲除小鼠的特征，以检测杂合子和纯合子中的任何表型变化。

项目成果

Glew R.H.and Ninomiya Y.: "Clinical Studies in Medical Biochemistry" Oxford University Press, 449 (1997)

Glew R.H. 和 Ninomiya Y.：“医学生物化学的临床研究”牛津大学出版社，449 (1997)

Ljubinov,AV: "Basemenet membrane abnormalities in human eye with diabetic retinopathy." J.Histochem.Cytochem.44. 1469-1479 (1996)

Ljubinov，AV：“患有糖尿病性视网膜病变的人眼基底膜异常。”

Oohashi T.: "Isolation and structure of genomic DNA encoding the human α6 (IV) collagen chain and comparison with other type IV collagen genes." J.Biol.Chem.270. 26863-26867 (1995)

Oohashi T.：“编码人类 α6 (IV) 胶原链的基因组 DNA 的分离和结构以及与其他 IV 型胶原基因的比较。J.Biol.Chem.270 (1995)。

Hino S: "Absence of α6(IV)collagen in kidney and skin of X-linked Alport syndrome." Pediatric Nephrology. 10. 742-744 (1996)

Hino S：“X 连锁 Alport 综合征的肾脏和皮肤中缺乏 α6(IV) 胶原。” 10. 742-744 (1996)

Ljubimov A.V.: "Extracellular alterations in human corneas with bullous keratopathy." Invest.Ophthal mol.Vis.Sci.37. 997-1007 (1996)

Ljubimov A.V.：“患有大疱性角膜病的人类角膜的细胞外改变。”