A Cancer Rainbow Mouse for Simultaneous Assessment of Multiple Oncogenes

 描述（由申请人提供）：用于同时评估多种癌基因的癌症彩虹小鼠癌症基因工程小鼠模型对于研究肿瘤形成的基本细胞和分子机制以及随后评估前瞻性治疗具有重要意义。然而，有几个因素限制了当前模型的成功和下一代技术的发展。首先，概念化、工程化和新小鼠品系的产生超出了大多数实验室的范围。这些项目的失败率很高，即使是最有经验和声望的研究项目也会继续面临重大风险。其次，在体内确定肿瘤驱动基因在重要细胞行为（如分化、增殖和迁移）中的作用通常需要广泛的化合物育种，这使得这些实验昂贵且耗时。第三，深度测序继续以一种速度识别新的肿瘤驱动基因，其中每个小鼠范式使用一个驱动基因无法跟上。因此，为了满足这些苛刻的需求，我们开发了Cancer rainbow（Crainbow）小鼠平台，以在单个遗传学上易于处理的系统中生成用户定义的、可靠的和多路复用的肿瘤发生模型。在我们的R21 IMAT资助项目中，我们已经证明了这项技术的体内原理验证，现在已经超出了该项目的初始阶段。有了这个系统，我们能够在多种组织和细胞类型中随机表达多种肿瘤驱动基因，并使用光谱可分辨的荧光蛋白报告基因在单细胞水平上同时监测它们的作用。这项R33提案的目的是对我们的新兴技术进行广泛而严格的测试，以确保其可靠性和科学界的可访问性。为了实现这一目标，我们的具体目标将是：1）优化彩虹生成和表达，2）在体内优化彩虹技术，以及3）在体内优化驱动基因活性的定量多重分析。我们希望Crabow平台能够改变癌症研究，使所有研究人员能够按需可靠地构建癌症小鼠模型，并将固有风险降至最低。Crabow的灵活和前瞻性设计还将使基因组编辑工具的强大交付成为可能，以提供具有细胞和时间精确分辨率的内源性编辑驱动基因的多路复用。通过多重驱动基因分析和分散的用户基础，Crabow平台将能够对多种癌症进行协同建模，并为测试个性化治疗干预提供未来模型。

 DESCRIPTION (provided by applicant): A Cancer rainbow mouse for simultaneous assessment of multiple oncogenes Genetically engineered mouse models of cancer hold significant promise for studying the basic cellular and molecular mechanisms underlying tumor formation and then evaluating prospective therapies. However, several factors have limited the success of current models and development of next-generation technologies. First, the conceptualization, engineering, and generation of a new mouse strain is outside the scope of most laboratories. These projects encounter high rates of failure and continue to carry significant risk for even the most seasoned and prestigious research programs. Second, establishing roles for tumor driver genes in vivo in important cellular behaviors such as differentiation, proliferation, and migration often requires extensive compound breeding, making these experiments expensive and time consuming. Third, deep-sequencing continues to identify new tumor driver genes at a rate in which the utilization of a one driver gene per mouse paradigm is unable to keep pace. Therefore to meet these demanding needs, we have developed the Cancer rainbow (Crainbow) mouse platform to generate user-defined, reliable, and multiplexed models of tumorigenesis in a single genetically tractable system. In our R21 IMAT funded project, we have demonstrated in vivo proof-of-principle for this technology and are now beyond the initial phases of this project. With this system we are able to stochastically express multiple tumor driver genes in a diversity of tissues and cell-types and simultaneously monitor their effects at a single cell level using spectrally resolvable fluorescent protein reporters. The objective of this R33 proposal is to perform extensive and rigorous testing of our emerging technology to ensure its reliability and accessibility for the scientific community. To achieve this objective, our specific aims will 1) Optimize Crainbow Generation and Expression, 2) Validate Crainbow technology In Vivo, and 3) Validate Quantitative Multiplex Analysis of Driver Gene Activity In Vivo. We expect the Crainbow platform to transform cancer research by enabling all investigators to reliably build mouse models of cancer on-demand and with minimal inherent risk. The flexible and forward-thinking design of Crainbow will also enable robust delivery of genome-editing tools to provide multiplexing of endogenously edited driver genes with cellular and temporally precise resolution. Through multiplex driver gene analysis and enabling of a decentralized user-base, the Crainbow platform will enable synergistic modeling of a diversity of cancers and provide future models for testing personalized therapeutic intervention.