EXTRACELLULAR MATRIX GENE EXPRESSION IN CORNEA

角膜细胞外基质基因表达

• 批准号: 3264217
• 负责人: BJORN OLSEN
• 金额: $ 19.6万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-09-30 至 1995-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3264217
• 关键词: Fuchs' dystrophy; antibody specificity; athymic mouse; autosomal dominant trait; bacterial virus; basement membrane; biochemical evolution; collagen; corneal endothelium; electrophoresis; extracellular matrix; gene expression; genetic library; genetic manipulation; genetic regulation; genetic strain; genome; histochemistry /cytochemistry; histogenesis; human genetic material tag; human tissue; immunochemistry; immunoelectron microscopy; immunofluorescence technique; immunoprecipitation; in situ hybridization; laboratory mouse; laboratory rabbit; molecular cloning; monoclonal antibody; nucleic acid sequence; protein biosynthesis; proteins; radioassay; tissue /cell culture

The primary goals of the study proposed here are: 1) to examine the molecular organization of rabbit type VIII collagen, the major constituent of the Descemet's membrane, and to establish the structural and evolutionary relationship between the gene(s) encoding the alpha2(VIII) chain and the genes that code for the alpha1(VIII) and alpha1(X) chains; 2) to examine the synthesis, assembly and location of the type VIII collagen molecule in the highly organized extracellular matrices of the cornea and in extraocular tissues; 3) to isolate the human type VIII collagen genes and examine hereditary disorders that may be linked to abnormalities in type VIII collagen gene structure and/or expression; 4) to isolate the mouse alpha1(VIII) gene and disrupt the endogenous gene in embryonic stem cells by insertional mutagenesis, as a first step in developing mouse strains with defects in the expression of type VIII collagen. To accomplish these goals, we will use a combination of DNA cloning/sequencing, protein chemistry and immunologic techniques. cDNA coding for the alpha2(VIII) chain will be synthesized from rabbit corneal endothelial cell mRNA. The gene coding for the alpha2(VIII) chain will be isolated from rabbit genomic libraries. Specific antibodies against synthetic peptides deduced from the nucleotide sequence of rabbit cDNAs will be generated and utilized to examine the synthesis, assembly and the location of the type VIII collagen in adult cornea as well as in corneal development. The primary structure of human type VIII collagen will be determined by isolation and sequencing of human genomic DNA encoding alpha1(VIII) collagen and specific antibodies will be generated against synthetic peptides. We also propose to examine whether the accumulation of collagen posterior to Descemet's membrane observed in autosomal dominantly inherited Fuchs' dystrophy and/or cornea guttata is due to altered structure and/or expression of type VIII collagen genes. Finally, to provide a basis for studies of the consequences of alterations in alpha1(VIII) collagen gene structure and expression in an animal model, we propose to disrupt the endogenous gene by homologous recombination in mouse embryonic stem cells. If successful, these experiments may allow generation of transgenic mice that could be models of hereditary disorders of type VIII collagen.

这里提出的研究的主要目标是：1）检查 兔 VIII 型胶原蛋白的分子组织，主要成分 后弹力层的膜，并建立结构和 编码 alpha2(VIII) 的基因之间的进化关系 链以及编码 alpha1(VIII) 和 alpha1(X) 链的基因； 2） 检查 VIII 型胶原蛋白的合成、组装和位置 角膜高度组织的细胞外基质中的分子 在眼外组织中； 3）分离人类VIII型胶原蛋白基因 并检查可能与异常相关的遗传性疾病 VIII型胶原蛋白基因结构和/或表达； 4）隔离 小鼠α1(VIII)基因并破坏胚胎干中的内源基因 通过插入诱变细胞，作为发育小鼠的第一步 VIII 型胶原蛋白表达缺陷的菌株。为了完成 为了实现这些目标，我们将结合使用 DNA 克隆/测序、蛋白质 化学和免疫学技术。 编码 alpha2(VIII) 链的 cDNA 将从兔体内合成 角膜内皮细胞 mRNA。编码 alpha2(VIII) 链的基因 将从兔基因组文库中分离出来。特异性抗体 从兔 cD​​NA 的核苷酸序列推导出的合成肽 将生成并用于检查合成、组装和 VIII 型胶原蛋白在成人角膜以及角膜中的位置 发展。人类 VIII 型胶原蛋白的主要结构是 通过人类基因组 DNA 编码的分离和测序来确定 α1(VIII) 胶原蛋白和特异性抗体将产生 合成肽。我们还建议检查是否积累了 常染色体显性观察到后弹力层后胶原 遗传性福克斯营养不良和/或角膜点滴是由于改变 VIII 型胶原蛋白基因的结构和/或表达。最后，为了 为研究变化的后果提供基础 α1(VIII) 胶原蛋白基因结构和在动物模型中的表达，我们 提出通过同源重组破坏小鼠的内源基因 胚胎干细胞。如果成功的话，这些实验可能会产生 可能成为遗传性疾病模型的转基因小鼠 VIII 胶原蛋白。