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

基本信息

批准号：
10558663
负责人：
Amjad Askary
金额：
$ 24.9万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-30 至 2024-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10558663
关键词：
Address Affect Area Award Bar Codes Biology Biomedical Engineering Birth Blindness Brain Cell Lineage Cells Chromatin Clustered Regularly Interspaced Short Palindromic Repeats Collaborations Competence Core Facility Development Developmental Biology Disease Engineering Epigenetic Process Future Gene Expression Profile Gene Expression Profiling Generations 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 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 editor career cell fate specification cell type cellular development design epigenetic regulation experimental study genome editing graduate student in situ imaging insight meetings member neurogenesis new technology novel organizational structure preservation progenitor programs reconstruction retinal neuron retinal progenitor cell retinal regeneration skills success synthetic biology visual information visual processing

项目摘要

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.

抽象的该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，该提案重点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来靶向合成条形码阵列中的突变可以永久且可遗传地记录个体的历史细胞的基因组中。重要的是，这里开发的方法与基于成像的读数兼容信息，并可以与原位转录分析相结合，提供当前的统一视图单个细胞的状态、过去的历史和空间背景。通过应用这些方法，我将绘制克隆图谱小鼠视网膜的结构，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，并重建视网膜的单细胞轨迹祖细胞经历不同的能力状态。这些实验共同提供了对哺乳动物视网膜细胞命运规范过程的基本新见解，因此将有助于再生医学的未来努力是为了纠正视网膜的错误指定或退化的情况神经元导致失明。我的职业目标是经营一个学术实验室，研究细胞如何在生命过程中获得和维持不同的身份。使用分子记录和合成生物学方法进行开发。拟议的实验将为我提供视网膜生物学、多重序列 FISH 技术和单细胞方面的进一步培训该目标所需的基因组学。我和我的导师迈克尔博士制定了详细的培训计划 Elowitz，帮助我过渡到独立。我将定期与 Elowitz 博士会面讨论研究进展、资助写作策略、学生指导和实验室管理。实践我的指导技能方面，我将监督实验室中研究生和技术人员的工作。为了拓宽我的科学视野通过网络，我每年将在 2-3 个国际会议上展示我的工作。为了寻求额外的指导，对于该项目成功至关重要的领域，我将与 Connie Cepko 博士合作并接受其指导，视网膜生物学专家、序贯 FISH 方法先驱蔡龙博士和视网膜生物学专家 Jay Shendure 博士单细胞基因组学。作为加州理工学院生物与生物工程系的成员，我将接触合成生物学和发育生物学领域的领导者、尖端课程和培训计划，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项雄心勃勃的研究计划，重建人类的发展历史哺乳动物的视网膜。