Biological function of basement membrane

1)克隆了小鼠α 6 （IV）胶原蛋白链的多个重叠cdna，确定了α 6 （IV）胶原蛋白链多肽的初级结构。2)从小鼠α 6 （IV）胶原蛋白多肽链各部分提取氨基酸序列，制备单克隆抗体，用于组织分布。结果表明，该链的组织分布与人α 6 （IV）胶原链相似。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是完全同步的。3)我们有一些迹象表明外显子31有选择性剪接。目前，我们正在从组织特异性表达的角度来描述这种非典型剪接事件。4)为了构建col4a6基因敲除小鼠，我们构建了几种结构，包括取出外显子2并插入外源基因Neo^R基因。通过同源重组将构建体转染到干细胞中。目前，我们正在对敲除小鼠进行表征，以检测杂合子和纯合子的表型变化。

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.