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 α6 (IV) 胶原链的重叠 cDNA，并确定了 α6 (IV) 链多肽的一级结构。 2) 我们针对小鼠 α6 (IV) 各个部分的氨基酸序列产生了单克隆抗体）胶原蛋白多肽链并将其用于组织分布。结果表明，该链的组织分布与人类 α6 (IV) 胶原链的组织分布相似。有趣的是，配对基因的分布；除了肾小球基底膜外，col4a1/col4a2、col4a3/col4a4 和 col4a5/col4a6 非常同步。3) 我们有一些迹象表明外显子 31 中存在选择性剪接。目前，我们正在根据组织特异性来表征这种非典型剪接事件4) 为了创建 col4a6 敲除小鼠，我们制作了几种构建体，其中包括取了外显子 2 的构建体并插入外源基因 Neo^R 基因。通过同源重组将构建体转染至干细胞中。目前，我们正在对基因敲除小鼠进行表征，以检测杂合子和纯合子的任何表型变化。

项目成果

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)。

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。”

Rauf S.: "Nephritogenicity and a-chain composition of NCl fractions of type IV collagen from bovine renal basement membrane." Virchow Archiv. 428. 281-288 (1996)

Rauf S.：“牛肾基底膜 IV 型胶原 NCl 部分的肾致炎性和 a 链组成。”

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。”

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)