MOLECULAR DETERMINANTS OF CYTOSKELETAL ORGANIZATION

细胞骨架组织的分子决定因素

• 批准号: 3292053
• 负责人: JONATHAN G IZANT
• 金额: $ 13.18万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 1991-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3292053
• 关键词: cytoskeleton; developmental genetics; eukaryote; gene expression; gene mutation; gene therapy; genetic library; genetic manipulation; genetic recombination; messenger RNA; microinjections; molecular cloning; nonmammalian vertebrate embryology; plasmids; tissue /cell culture

Cytoskeletal architecture is a predominant and characteristic feature of eukaryotic cell identity and function. The goal of this project is to study the activity of genes that coordinate cytoskeletal organization in cultured cells and amphibian embryos in vivo using recombinant DNA vectors including anti-sense plasmids. Anti-sense gene inhibition is a valuable molecular strategy for phenocopying gene mutations in vertebrate organisms that are not amenable to classical genetic analysis. Studies on the mechanism of DNA mediated "flipped" gene anti-sense inhibition will continue. Anti-sense chicken thymidine kinase intron and anti-sense intron-exon junction plasmids will be constructed and tested by comicroinjection with the wild type gene into cultured cell nuclei and Xenopus oocyte germinal vesicles. Gene product activity and target mRNA transcript metabolism will be carefully monitored. Related strategies for gene replacement will be explored. Anti-sense and sense plasmids containing several cytoskeleton-associated protein genes (tropomyosins, yield-actinin, and a 200 kd microtubule-associated protein) will be constructed and introduced into cultured cells and Xenopus embryos. The resultant deficiencies in cellular architecture will be studied to assess the contribution that each gene makes to cellular morphology, viral transformation, and embryonic morphogenesis. Xenopus cDNA library inserts, enriched in embryo-specific sequences, will be subcloned into anti-sense expression vectors and then introduced into Xenopus embryos to identify novel genes that direct embryonic pattern formation. The objective is to use anti-sense gene inhibition as a pseudo-genetic tool to identify previously uncharacterized factors that regulated cell organization in addition to using these molecular approaches for the analysis of cloned gene function in living cells.

细胞骨架结构是细胞骨架的一个主要特征 真核细胞的身份和功能。 该项目的目标是 研究协调细胞骨架组织的基因的活性 使用重组 DNA 载体在体内培养细胞和两栖动物胚胎 包括反义质粒。 反义基因抑制是一种有价值的分子策略 脊椎动物中不适合的表型基因突变 到经典的遗传分析。 DNA介导的机制研究 “翻转”的基因反义抑制将继续。 反义鸡 胸苷激酶内含子和反义内含子-外显子连接质粒将 通过将野生型基因注射到体内来构建和测试 培养的细胞核和非洲爪蟾卵母细胞生发囊泡。 基因产物 活性和目标 mRNA 转录代谢将被仔细研究 被监控。 将探索基因替换的相关策略。 含有多个细胞骨架相关的反义和正义质粒 蛋白质基因（原肌球蛋白、产量肌动蛋白和 200 kd 微管相关蛋白）将被构建并引入 培养细胞和非洲爪蟾胚胎。 由此产生的细胞缺陷 将研究架构以评估每个基因的贡献 影响细胞形态、病毒转化和胚胎 形态发生。 非洲爪蟾 cDNA 文库插入片段富含胚胎特异性序列，将 亚克隆到反义表达载体中，然后引入 非洲爪蟾胚胎识别指导胚胎模式的新基因 形成。 目的是利用反义基因抑制作为 伪遗传工具来识别以前未表征的因素 除了使用这些分子方法之外，还可以调节细胞组织 用于分析活细胞中克隆基因的功能。