EXTRACELLULAR MATRIX GENE EXPRESSION IN CORNEA

角膜细胞外基质基因表达

• 批准号: 3264219
• 负责人: BJORN OLSEN
• 金额: $ 20.05万
• 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/3264219
• 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)检查