Next-generation mouse gene-targeting technology to model tumorigenesis

下一代小鼠基因靶向技术模拟肿瘤发生

• 批准号: 8621211
• 负责人: Zhenghe Wang
• 金额: $ 24.13万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-23 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8621211
• 关键词: Acute Myelocytic Leukemia; Alleles; Antineoplastic Agents; Archives; Automobile Driving; Biology; Cancer Biology; Carcinoma; Clear Cell; Communities; DAXX gene; Development; Embryo; Failure; Frequencies; Gene Targeting; Generations; Genes; Genetically Engineered Mouse; Genome; Germ Lines; Goals; Hot Spot; Human; Individual; Knock-in Mouse; Knock-out; Knockout Mice; Malignant Neoplasms; Mediating; Methods; Microinjections; Modeling; Mouse Strains; Mus; Mutant Strains Mice; Mutate; Mutation; Neuroendocrine Tumors; Oncogenic; Ovarian; Pancreas; Pancreatic Cyst; Phenotype; Process; Production; Recombinant adeno-associated virus (rAAV); Relative (related person); Role; Somatic Mutation; Speed; System; Technology; The Cancer Genome Atlas; Tumor Suppressor Genes; Tumor-Derived; anticancer research; cancer genetics; cost effective; drug development; homologous recombination; innovation; innovative technologies; mouse development; mouse model; mutant; next generation; novel; nuclease; oligodendroglioma; prevent; public health relevance; repository; technology development; tool; tumor; tumorigenesis; zygote

Abstract The goal of this application is to develop a very fast and cost-effective method to generate gene-targeted mice to model tumorigenesis. Gene-targeted mice are invaluable tools to determine the roles of oncogenic mutations in cancer development. However, conventional gene targeting is slow, expensive and prone to failure. While nuclease-mediated targeting may speed the production of mutants, there remain significant concerns about off-target mutations, relative ease of use and access to the entire genome. In preliminary studies, we have successfully developed an innovative method to directly and efficiently target mouse fertilized eggs using recombinant adeno-associated virus (rAAV)-mediated homologous recombination. Using this approach, we were able to generated germ-line-transmitting mice with at least 10% targeting frequency in a month. We believe that our technology is superior to nuclease-mediated gene targeting approaches (e.g. ZFN, TALEN and CRISPR/Cas). In contrast to nuclease-mediated approaches, off-target mutations are infrequent, embryos can be processed en masse without individual microinjection, and all regions of the genome are accessible to manipulation. Here we propose to further develop this technology to generate gene-targeted mice to model tumorigenesis by determining: (a) if gene-targeted mice generated by our method are suitable for modeling tumorigenesis, (b) if our approach is generally applicable to create gene-targeted mice of various tumor suppressors and oncogenes, and (c) if our approach can be used to generate conditional knock-out and knock-in mice. Successful development of these technologies will revolutionize generation of genetically engineered mice to model tumorigenesis. It will have huge impacts on basic cancer biology as well as cancer drug development.

抽象的 该应用程序的目的是开发一种非常快速且具有成本效益的方法来产生靶向基因的小鼠 建模肿瘤发生。靶向基因的小鼠是确定致癌作用的宝贵工具 癌症发展中的突变。但是，传统的基因靶向很慢，昂贵且容易 失败。虽然核酸酶介导的靶向可能会加快突变体的产生，但仍然存在显着 担心脱靶突变，相对易于使用和访问整个基因组。在初步 研究，我们成功地开发了一种创新的方法来直接有效地靶向小鼠受精 使用重组腺相关病毒（RAAV）介导的同源重组的卵。使用此 方法，我们能够在A中产生具有至少10％靶向频率的种系传输小鼠 月。我们认为，我们的技术优于核酸酶介导的基因靶向方法（例如ZFN，， Talen和CRISPR/CAS）。与核酸酶介导的方法相反，脱靶突变很少见 可以在没有单独的显微注射的情况下对胚胎进行处理，并且基因组的所有区域均为 可以操纵。在这里，我们建议进一步开发这项技术以生成靶向基因 通过确定肿瘤发生的小鼠：（a）如果我们的方法产生的基因靶向小鼠是合适的 用于建模肿瘤发生，（b）如果我们的方法通常适用于创建靶向基因的小鼠 肿瘤抑制剂和癌基因，以及（c）如果我们的方法可用于产生有条件的敲除和 敲入小鼠。这些技术的成功发展将彻底改变基因的产生 工程小鼠以建模肿瘤发生。这将对基本癌症生物学和癌症产生巨大影响 药物开发。