Mendelian inheritance of artificial chromosomes

合成哺乳动物人工染色体(Mac)代表了基因组技术的一个新前沿， 有可能改变染色体和合成生物学，并刺激无数 医学上的突破性进展。人类基因组计划-WRITE旨在产生一套完整的人造人类 染色体。如果不能实现这一雄心勃勃的目标，Mac在生物技术方面有巨大的突破潜力。 以及医学，例如为药物开发或采集患者创造人性化的动物模型- 个人化器官移植。此外，从最少的组件构建Mac将会取得进展 我们对哺乳动物染色体组成的基本理解。 作为遗传的载体，功能齐全的染色体通过 有丝分裂与真核有性生殖和孟德尔的减数分裂 继承。我们的目标是构建第一个通过生殖系实现忠实遗传的Mac，使用 以鼠标为模型系统。一个障碍是着丝粒，即每条染色体上指向 通过有丝分裂和减数分裂进行传播。因为哺乳动物着丝粒不是在DNA中编码的 目前尚不清楚如何构建包含这一关键元件的合成染色体。确实有 在减数分裂中创造作为同源染色体配对和重组的Mac的额外挑战。至 解决这些问题，我们将劫持现有的细胞机器来组装着丝粒染色质和 加入更多的遗传因素，以确保减数分裂配对和重组。 这项工作需要在多个层面上进行创新：设计编码关键函数的MAC向量 元素，组装大型合成DNA结构，并最终创造出在体内测试Mac的动物。 拟议的工作建立在联合调查组在所有这些领域取得的最新进展的基础上，我们 有关键的工具和专业知识来构建必要的DNA模板，将它们引入胚胎， 分析结果，并根据需要制定替代战略。最有意义的初步数据 将显示一个人造染色体，它通过有丝分裂和减数分裂成功地传播 但实现这一步是我们建议的一个主要目标，需要大量的时间投入 和努力。因此，我们请求在没有初步数据的情况下为这个项目提供支持 成功的可能性很高，有理由考虑将我们的建议作为T-R01机制的一部分。 我们使用小鼠作为一个相对快速和易处理的哺乳动物模型系统，具有出色的 测试和调试Mac的机会，我们的进步应该很容易转移到其他物种 在生物技术和医学方面的应用。这个项目的成功将代表着在 开发在体内具有全功能的人造染色体，提供前所未有的 在动物模型中的基因组工程能力，并使不同的合成生物学应用。

Synthetic mammalian artificial chromosomes (MACs) represent a new frontier in genome technology, with the potential to transform chromosome and synthetic biology and stimulate the development of numerous radical advances in medicine. Human Genome Project-Write aims to generate an entire set of synthetic human chromosomes. Short of this ambitious goal, MACs have enormous potential for breakthroughs in biotechnology and medicine, such as creating humanized animal models for drug development or for harvesting patient- personalized organs for transplantation. Furthermore, building MACs from minimal components will advance our fundamental understanding of what comprises a mammalian chromosome. As vehicles for genetic inheritance, fully functional chromosomes are faithfully transmitted through mitosis and the specialized meiotic divisions underlying eukaryotic sexual reproduction and Mendelian inheritance. Our goal is to construct the first MACs that achieve faithful inheritance through the germline, using mouse as a model system. One obstacle is the centromere, the locus on each chromosome that directs transmission through both mitosis and meiosis. Because mammalian centromeres are not encoded in the DNA sequence, it is unclear how to build synthetic chromosomes containing this crucial element. There are additional challenges to create MACs that pair and recombine as homologous chromosomes in meiosis. To solve these problems, we will hijack the existing cellular machinery for assembling centromere chromatin and incorporate additional genetic elements to ensure meiotic pairing and recombination. This effort requires innovation at multiple levels: designing MAC vectors encoding key functional elements, assembling large synthetic DNA constructs, and ultimately creating animals to test MACs in vivo. The proposed work builds on recent advances from the co-investigators’ teams in all of these areas, and we have key tools and expertise in place to build the necessary DNA templates, introduce them into embryos, analyze the outcomes, and develop alternative strategies as necessary. The most meaningful preliminary data would be to show a synthetic artificial chromosome that is successfully transmitted through mitosis and meiosis in vivo, but achieving this step is a major goal of our proposal and will require substantial investment of time and effort. Thus, we are requesting support for this project without the preliminary data that would demonstrate high likelihood of success, justifying consideration of our proposal as part of the T-R01 mechanism. We use mouse as a relatively rapid and tractable mammalian model system with outstanding opportunities for testing and debugging MACs, and our advances should readily transfer to other species for applications in biotechnology and medicine. Success in this project will represent a quantum leap in the development of synthetic artificial chromosome that are fully functional in vivo, providing unprecedented genome engineering capabilities in animal models and enabling diverse synthetic biology applications.