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Use of RecA to Promote Gene Targeting

使用 RecA 促进基因靶向

基本信息

批准号：
7786194
负责人：
Jeffrey J Essner
金额：
$ 18万
依托单位：
IOWA STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-03-01 至 2012-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7786194
关键词：
Address Agriculture Animal Model Animals Biological Models Cells Chimeric Proteins Cloning DNA DNA Binding Domain Development Double Strand Break Repair Embryo Engineering Enzymes Eukaryota Event Filament Frequencies Gene Conversion Gene Targeting Generations Genes Genetic Genetic Complementation Test Genetic Recombination Genetic Screening Genome Genomics Goals Grant Head Health Homologous Gene Human Human Genome Induced Mutation Injection of therapeutic agent Laboratories Location Mediating Methods Microinjections Mitotic Modeling Modification Molecular Molecular Biology Mutate Mutation Names Nature Nuclear Localization Signal Optics Pathway interactions Pharmacologic Substance Principal Investigator Probability Production Proteins Rec A Recombinases Reporter Genes Role Scientist Single-Stranded DNA Site Site-Directed Mutagenesis Specific qualifier value System Techniques Technology Testing Work Zebrafish Zinc Fingers base cost design developmental genetics drug discovery embryo cell embryonic stem cell experience gene therapy human disease insertion/deletion mutation interest mutant next generation nuclease positional cloning programs protein purification public health relevance recombinase recombinational repair repaired research study skills success tool zebrafish genome

项目摘要

DESCRIPTION (provided by applicant): The forward genetic screens conducted in zebrafish have firmly established this system as a model for human disease and development. Sequencing of the zebrafish genome and the development of powerful genomics-based tools have also contributed to the placement of this model system at the forefront of vertebrate developmental genetics. Despite this prominent role for zebrafish, the ability to fully utilize genomics- based tools in this model system is limited by the inability to perform reverse genetics. To address this need, we propose to develop simple methods that most labs with moderate molecular biology skills could use to disrupt or target genes. Our method uses a significantly modified version of the bacterial recombinase protein RecA that has greatly enhanced activities. RecA is a multifunctional enzyme involved in recombination and the repair of double-strand breaks. RecA has diverse enzymatic functions, including the ability to coat single-stranded DNA, to carry out homology searches, and to promote strand exchange during recombination. We propose to use our modified version of RecA to coat single stranded DNA and produce DNA-RecA filaments that are homologous to the gene we wish to target. In Aim 1, we will test whether these filaments can create small deletions and insertions in the zebrafish genome. In Aim 2, we will determine the frequency with which the single-stranded DNA-RecA filaments promote targeted insertion of a reporter gene into the zebrafish genome. Both of the activities described in the above two aims would be consistent with the ability of the single- stranded DNA-RecA filaments to create double strand breaks. We provide significant preliminary results to support both of these aims and demonstrate how injection of chimeric versions of RecA proteins can mediate gene targeting in mitotic cells of the early zebrafish embryo and in the zebrafish germline. While these techniques are being developed in zebrafish, we believe they can be adapted for genome modifications in most models for human disease and will have applications for human gene therapy. PUBLIC HEALTH RELEVANCE: We propose to develop methods in zebrafish that utilize the bacterial recombinase protein RecA to create site directed modification of the genome. These techniques have the potential to revolutionize reverse genetic approaches in all animal models of human disease. Successful completion of this proposal will have a profound impact on the identification of pharmaceutical targets for drug discovery and the production of new animal models of human disease.

描述（由申请人提供）：在斑马鱼中进行的正向遗传筛选已牢固地将该系统确立为人类疾病和发育的模型。斑马鱼基因组的测序和强大的基于基因组学的工具的开发也有助于将该模型系统置于脊椎动物发育遗传学的前沿。尽管斑马鱼发挥着重要作用，但在该模型系统中充分利用基于基因组学的工具的能力由于无法进行反向遗传学而受到限制。为了满足这一需求，我们建议开发简单的方法，大多数具有中等分子生物学技能的实验室都可以使用这些方法来破坏或靶向基因。我们的方法使用了细菌重组酶蛋白 RecA 的显着修改版本，其活性大大增强。 RecA 是一种多功能酶，参与重组和双链断裂修复。 RecA 具有多种酶功能，包括包被单链 DNA、进行同源性搜索以及在重组过程中促进链交换的能力。我们建议使用我们的 RecA 修饰版本来包被单链 DNA 并产生与我们希望靶向的基因同源的 DNA-RecA 丝。在目标 1 中，我们将测试这些细丝是否可以在斑马鱼基因组中产生小的缺失和插入。在目标 2 中，我们将确定单链 DNA-RecA 丝促进报告基因定向插入斑马鱼基因组的频率。上述两个目标中描述的两种活性都与单链 DNA-RecA 丝产生双链断裂的能力一致。我们提供了重要的初步结果来支持这两个目标，并证明注射嵌合版本的 RecA 蛋白如何介导早期斑马鱼胚胎有丝分裂细胞和斑马鱼种系中的基因靶向。虽然这些技术正在斑马鱼中开发，但我们相信它们可以适用于大多数人类疾病模型中的基因组修饰，并将应用于人类基因治疗。公共健康相关性：我们建议开发斑马鱼方法，利用细菌重组酶蛋白 RecA 对基因组进行定点修饰。这些技术有可能彻底改变人类疾病所有动物模型中的反向遗传方法。该提案的成功完成将对药物发现的药物靶标的确定和人类疾病新动物模型的产生产生深远的影响。