Gene Targeting Using Modular Zinc Finger Nulceases

使用模块化锌指核酸酶进行基因靶向

• 批准号: 8100644
• 负责人: HOWARD I SIROTKIN
• 金额: $ 7.85万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8100644
• 关键词: Alleles; Animal Model; Binding; Binding Sites; Biological Models; Biological Process; Biomedical Research; Cell Culture System; Chimeric Proteins; Computer Assisted; DNA Binding Domain; DNA Double Strand Break; Effectiveness; Embryo; Engineering; Exonuclease; Gene Targeting; Genes; Genetic; Germ-Line Mutation; Goals; Human Genome Project; Laboratories; Lesion; Mammalian Cell; Mammals; Mediating; Methods; Molecular; Monitor; Mutate; Mutation; Plants; Procedures; Protocols documentation; Reporting; Research Personnel; System; Technology; Testing; Zebrafish; Zinc Fingers; biological systems; cost; design; gene function; interest; loss of function; novel; nuclease; research study; tool

DESCRIPTION (Provided by Applicant): One fundamental method to investigate biological processes is by loss-of-function studies. The goal of these efforts is to determine the activity of a gene by disrupting that gene and monitoring the effects on a biological system. Recently, gene targeting technology has been developed using zinc finger nucleases (ZFNs) as a method to mutate specific genes. The approach involves engineering zinc finger DNA binding domains to recognize specific target sequences. These domains can then be tethered to the FOK1 exonuclease domains to generate a fusion protein that can induce double stranded DNA breaks near the zinc finger binding sites. ZFNs have been successfully used to disrupted genes in a variety of systems ranging from plants to mammals. Thus far, only a relatively small number of laboratories have reported successful efforts to employ ZFNs. The use of ZFNs has been curtailed by the difficulty in generating zinc finger domains that bind to specific target sequences, and by the high cost of commercially available ZFNs. In preliminary studies, computer aided "modular" design has been an effective means to engineer functional ZFNs. The experiments in this application evaluate the effectiveness of a strategy to use modular ZFN design to generate germ-line mutations using zebrafish as a model system. Zebrafish is a powerful system for genetic, cellular, and molecular studies, and the ability to routinely perform gene targeting studies would be a major advance. The methods in this application are broadly applicable to biomedical research in both cell culture systems and model organisms. RELEVANCE: The human genome project has identified thousands of novel genes, but the activity of many of these genes remains unknown. The ability to disrupt specific genes is a powerful means to determine the function of these genes. The experiments in this application evaluate the effectiveness of one means to accomplish this goal using model organisms and cell culture systems.

描述（由申请人提供）：研究生物过程的一种基本方法是功能丧失研究。 这些努力的目标是通过破坏基因并监测其对生物系统的影响来确定基因的活性。 最近，已经开发出使用锌指核酸酶（ZFN）作为突变特定基因的方法的基因打靶技术。 该方法涉及改造锌指 DNA 结合域以识别特定的靶序列。 然后，这些结构域可以与 FOK1 核酸外切酶结构域连接，生成融合蛋白，该融合蛋白可以在锌指结合位点附近诱导双链 DNA 断裂。 ZFN 已成功用于破坏从植物到哺乳动物等多种系统中的基因。 到目前为止，只有相对少数的实验室报告了采用 ZFN 的成功努力。 由于难以生成与特定靶序列结合的锌指结构域以及市售 ZFN 的高成本，ZFN 的使用受到限制。 在初步研究中，计算机辅助“模块化”设计已成为设计功能性 ZFN 的有效手段。 本应用中的实验评估了使用模块化 ZFN 设计以斑马鱼作为模型系统生成种系突变的策略的有效性。 斑马鱼是遗传、细胞和分子研究的强大系统，常规进行基因靶向研究的能力将是一个重大进步。 本申请中的方法广泛适用于细胞培养系统和模型生物体的生物医学研究。 相关性：人类基因组计划已鉴定出数千个新基因，但其中许多基因的活性仍然未知。 破坏特定基因的能力是确定这些基因功能的有力手段。 本申请中的实验评估了使用模型生物和细胞培养系统实现这一目标的一种方法的有效性。