EXTRACELLULAR MATRIX GENE EXPRESSION IN CORNEA

角膜细胞外基质基因表达

• 批准号: 3264218
• 负责人: BJORN OLSEN
• 金额: $ 19.21万
• 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/3264218
• 关键词: Fuchs' dystrophy; antibody specificity; athymic mouse; autosomal dominant trait; bacterial virus; basement membrane; biochemical evolution; collagen; corneal endothelium; electrophoresis; embryonic stem cell; 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)研究 兔VIII型胶原主要成分的分子结构 Descemet的膜，并建立结构和 编码α2的基因(S)之间的进化关系(Viii) 链以及编码alpha1(Viii)和alpha1(X)链的基因； 检测VIII型胶原的合成、组装和定位 分子在高度组织的角膜细胞外基质和 3)分离人型胶原基因 并检查遗传性疾病，这些疾病可能与 III型胶原基因结构和/或表达；4)分离 小鼠α1(VIII)基因与胚胎干细胞内源性基因的干扰 通过插入突变获得细胞，作为发育小鼠的第一步 有VIII型胶原表达缺陷的菌株。要完成 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。 将从兔体内合成编码α2(Viii)链的基因 角膜内皮细胞基因的表达。编码α2(Viii)链的基因 将从兔基因组文库中分离得到。特异性抗体 由兔cDNA核苷酸序列推导的合成肽 将被生成并用于检查合成、组装和 型胶原在成人角膜及角膜中的定位 发展。人类VIII型胶原的一级结构将是 由人类基因组编码DNA的分离和测序确定 将产生针对甲型()胶原蛋白和特异性抗体 合成肽。我们还建议研究是否积累了 常染色体显性分布的Descemet膜后胶原 遗传性Fuchs营养不良和/或角膜点滴是由于改变 III型胶原基因的结构和/或表达。最后，为了 为研究变化的后果提供了基础 α1(VIII)胶原基因结构及其在动物模型中的表达 提出通过同源重组打乱小鼠内源性基因 胚胎干细胞。如果成功，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型胶原蛋白。