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.

摘要 这项申请的目标是开发一种非常快速和具有成本效益的方法来产生基因靶向小鼠 来模拟肿瘤发生。基因靶向小鼠是确定致癌基因的作用的宝贵工具。 癌症发展中的突变。然而，传统的基因靶向是缓慢的、昂贵的并且容易发生突变。 失败虽然核酸酶介导的靶向可以加速突变体的产生，但仍然存在显著的缺陷。 对脱靶突变、相对容易使用和获得整个基因组的担忧。初步 研究，我们已经成功地开发出一种创新的方法，直接和有效地针对小鼠受精 使用重组腺相关病毒（rAAV）介导的同源重组的鸡蛋。使用此 通过这种方法，我们能够产生生殖系传播小鼠，在一个实验中至少有10%的靶向频率。 月我们相信我们的技术上级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，脱靶突变很少发生， 胚胎可以在没有单独显微注射的情况下进行连续处理，并且基因组的所有区域都是 容易被操纵在这里，我们建议进一步发展这项技术，以产生基因靶向 通过确定：（a）通过我们的方法产生的基因靶向小鼠是否适合 对于建模肿瘤发生，（B）如果我们的方法通常适用于产生各种基因靶向小鼠， 肿瘤抑制基因和癌基因，以及（c）我们的方法是否可以用于产生条件性敲除， 基因敲入小鼠这些技术的成功开发将彻底改变一代基因 改造小鼠来模拟肿瘤发生。它将对基本的癌症生物学以及癌症产生巨大的影响 药物开发