Deep cell history tracking: engineering cells that write their detailed life stories into their DNA to study DNA damage

深度细胞历史追踪：工程细胞将详细的生命故事写入 DNA 中以研究 DNA 损伤

• 批准号: 10301626
• 负责人: Theresa Berens Loveless
• 金额: $ 10万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10301626
• 关键词: Affect; Animals; Architecture; Biological; Cell Cycle; Cell physiology; Cells; Characteristics; Collection; Cultured Cells; DNA; DNA Damage; DNA damage checkpoint; Development; Dose; Engineering; Event; Gene Proteins; Genome; Genome Stability; Insertion Mutation; Lead; Life; Long-Term Effects; Longitudinal Studies; Malignant Neoplasms; Mammals; Molecular; Monitor; Mutation; Nucleotides; Optics; Pharmaceutical Preparations; Pharmacotherapy; Phase; Phenotype; Physiological; Population; RNA; Recording of previous events; Records; Resistance; Role; Sampling; Signal Transduction; Techniques; Technology; Testing; Therapeutic; Time; Transgenic Mice; Work; Writing; Xenograft Model; Xenograft procedure; base; experience; experimental study; genetically modified cells; genomic locus; in vivo; interest; molecular phenotype; mouse model; new technology; novel strategies; patient derived xenograft model; promoter; response; single-cell RNA sequencing; tissue culture; tool; triple-negative invasive breast carcinoma

Project Summary DNA recording is a recently developed technology that allows cellular signaling events of interest to be followed over time without removing cells from their physiological context. During a DNA recording experiment, genetically engineered cells expressing the DNA recorder acquire mutations at a “recording locus” as the result of transient cellular events, without perturbing their normal phenotype. At the end of the experiment, once their eventual phenotype is known, cells can be collected and their histories determined by sequencing the recording locus. The work proposed here will lead to a DNA recorder that is not limited, as current technology is, to recording one or two cellular events per experiment, but can record dozens of events in parallel, allowing an unbiased examination of cell history in any context compatible with genetically engineered cells. In this project, the new DNA recorder will be used to study long-term effects for a cell of experiencing significant spontaneous DNA damage. Although DNA damage is very well-studied, it has not previously been possible to follow the rare cells that experience significant spontaneous damage, especially in an in vivo mammalian context. The new DNA recorder will be developed in stages: First, intracellular recording machinery, based on a pre-existing recorder, will be created for a small number of cellular events associated with the DNA damage response. Next, the fidelity of this new recording machinery will be extensively validated in cultured cells and cancer xenografts. Finally, a new recording architecture will be developed and validated, before being introduced into a transgenic mouse to identify how spontaneous DNA damage affects cell fate determination during normal development.

项目概要 DNA 记录是一项最近开发的技术，可以跟踪感兴趣的细胞信号事件 随着时间的推移，细胞不会脱离其生理环境。在 DNA 记录实验中，基因 表达 DNA 记录器的工程细胞在“记录位点”获得突变，这是短暂的结果 细胞事件，而不扰乱其正常表型。在实验结束时，一旦他们最终 表型已知，可以收集细胞并通过对记录基因座进行测序来确定它们的历史。 这里提出的工作将导致一种 DNA 记录器，它不像当前技术那样仅限于记录一个 或每个实验两个细胞事件，但可以并行记录数十个事件，从而允许公正的 在任何与基因工程细胞兼容的情况下检查细胞历史。在这个项目中，新 DNA记录仪将用于研究经历显着自发DNA的细胞的长期影响 损害。尽管 DNA 损伤已得到充分研究，但以前不可能追踪罕见细胞 经历显着的自发损伤，特别是在哺乳动物体内。新的DNA 记录器将分阶段开发：首先，基于现有记录器的细胞内记录机器， 将为与 DNA 损伤反应相关的少量细胞事件创建。接下来是保真度 这种新的记录机制将在培养细胞和癌症异种移植物中得到广泛验证。最后，一个 新的记录架构将被开发和验证，然后被引入转基因小鼠中 确定自发 DNA 损伤如何影响正常发育过程中的细胞命运决定。