Synthetic recording and in situ readout of cell lineage and molecular history in mammalian retina

哺乳动物视网膜细胞谱系和分子历史的综合记录和原位读出

• 批准号: 10040342
• 负责人: Amjad Askary
• 金额: $ 11.87万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-30 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10040342
• 关键词: Address; Affect; Area; Award; Bar Codes; Biological; Biology; Birth; Blindness; Brain; Cell Lineage; Cells; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Competence; Core Facility; Development; Developmental Biology; Disease; Engineering; Epigenetic Process; Fibrinogen; Future; Gene Expression Profiling; Generations; Genetic Transcription; Genome; Genomics; Goals; Grant; Heterogeneity; Image; In Situ; Individual; Inherited; International; Intrinsic factor; Knowledge; Label; Lead; Lentivirus Vector; Libraries; Light; Maps; Measurement; Mentors; Mentorship; Methods; Molecular; Mus; Mutation; Natural regeneration; Neuroglia; Neurons; Organism; Pathway interactions; Pattern; Pilot Projects; Process; Readability; Recording of previous events; Regenerative Medicine; Regulation; Research; Resources; Retina; Retinal Degeneration; Role; Running; Signal Transduction; Site; Stereotyping; Structure; Students; System; Technology; Time; Tissues; Training; Training Programs; Trees; Viral; Viral Genome; Vision; Visual; Work; Writing; base; career; cell fate specification; cell type; design; epigenetic regulation; experimental study; genome editing; graduate student; in situ imaging; insight; meetings; member; neurogenesis; new technology; novel; preservation; progenitor; programs; reconstruction; retinal neuron; retinal progenitor cell; skills; success; synthetic biology; visual information; visual processing

Abstract The long term goal of this project is to describe how the intricate structure of mammalian retina is made during development. To achieve this goal, this proposal focuses on developing novel molecular recording technologies that enable reconstruction of cell lineage and molecular history based on in situ endpoint measurements. These methods leverage recent advances in genome editing technology to make targeted mutations in synthetic barcode arrays that make a permanent and inheritable record of history of individual cells in their genome. Importantly, the methods developed here are compatible with imaging based readout of information and can be combined with in situ transcriptional profiling to provide a unified view of the current state, past history, and spatial context of individual cells. By applying these methods, I will map the clonal structure of the mouse retina, investigate the extent to which local signals affect cell fate decisions, reveal the stochastic versus pre-programmed aspects of retinal lineage, and reconstruct the single cell trajectory of retinal progenitor cells as they proceed through different competence states. Together, these experiments provide fundamental new insight into cell fate specification process in mammalian retina and, therefore, will aid the future efforts in regenerative medicine to remedy conditions in which misspecification or degeneration of retinal neurons lead to blindness. My career goal is to run an academic lab that studies how cells acquire and maintain different identities during development, using molecular recording and synthetic biology approaches. The proposed experiments will provide me with further training in retina biology, multiplexed sequential FISH technology, and single cell genomics that are required for this goal. I have developed a detailed training plan with my mentor, Dr. Michael Elowitz, to help me transition to independence. I will meet regularly with Dr. Elowitz to discuss research progress, strategies for grant writing, student mentorship and lab management. To practice my mentorship skills, I will oversee the work of a graduate student and a technician in the lab. To broaden my scientific network, I will present my work at 2-3 international meetings per year. To seek out additional mentorship, in areas critical to the success of this project, I will collaborate with and receive guidance from Dr. Connie Cepko, expert in retina biology, Dr. Long Cai, pioneer in sequential FISH methods, and Dr. Jay Shendure, expert in single cell genomics. As a member of Caltech Division of Biology and Biological Engineering, I will have access to leaders in synthetic as well as developmental biology, cutting-edge courses and training programs, and state-of-the-art core facilities. The Pathway to Independence Award will provide the time and resources required to initiate an ambitious research program on the reconstruction of developmental history of mammalian retina.

摘要 这个项目的长期目标是描述哺乳动物视网膜的复杂结构是如何在 发展为了实现这一目标，本提案的重点是开发新型分子记录 能够基于原位终点重建细胞谱系和分子历史的技术 测量.这些方法利用基因组编辑技术的最新进展， 在合成条形码阵列中的突变，使个人的历史的永久性和可遗传的记录， 基因组中的细胞。重要的是，这里开发的方法与基于成像的读出兼容， 信息，并且可以与原位转录谱分析相结合，以提供对当前转录水平的统一看法。 状态、过去的历史和单个细胞的空间背景。通过应用这些方法，我将绘制克隆 小鼠视网膜的结构，研究局部信号影响细胞命运决定的程度，揭示 视网膜谱系的随机与预编程方面，并重建视网膜细胞的单细胞轨迹。 祖细胞通过不同的能力状态。这些实验提供了 对哺乳动物视网膜中细胞命运指定过程的基本新见解，因此，将有助于 在再生医学中未来的努力，以补救其中视网膜的错误指定或变性的条件， 神经元导致失明。 我的职业目标是管理一个学术实验室，研究细胞在生长过程中如何获得和保持不同的身份。 开发，使用分子记录和合成生物学方法。拟议的实验将 为我提供视网膜生物学，多重顺序FISH技术和单细胞的进一步培训， 基因组学是实现这一目标所必需的。我已经和我的导师迈克尔博士制定了详细的培训计划 埃罗威茨帮我过渡到独立我会定期与埃洛维茨博士会面讨论研究 进展，赠款写作，学生指导和实验室管理的战略。来实践我的导师身份 技能，我将监督一个研究生和一个技术员在实验室的工作。为了拓宽我的科学 我将在每年2-3次的国际会议上介绍我的工作。为了寻求更多的指导，在 对于这个项目的成功至关重要的领域，我将与Connie Cepko博士合作并接受他的指导， 视网膜生物学专家，序列FISH方法的先驱Long Cai博士和 单细胞基因组学作为加州理工学院生物学和生物工程系的成员，我将拥有 接触合成生物学和发育生物学的领导者，前沿课程和培训计划， 和最先进的核心设施独立之路奖将提供时间和资源， 需要启动一个雄心勃勃的研究计划，重建发展历史， 哺乳动物视网膜