Studying immune development at single-cell resolution by DNA barcoding

通过 DNA 条形码研究单细胞分辨率的免疫发育

• 批准号: 9234225
• 负责人: Boris Reizis
• 金额: $ 25.43万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-12-08 至 2018-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9234225
• 关键词: Alleles; Animals; Bone Marrow; Cell Lineage; Cell Separation; Cell Transplantation; Cells; Clone Cells; DNA; DNA Sequence; DNA Transposons; DNA sequencing; Dendritic Cells; Development; Environment; Genetic Recombination; Genome; Hematopoietic stem cells; Homeostasis; Immune; Immune response; Immune system; In Vitro; Kinetics; Label; Libraries; Morphologic artifacts; Mouse Strains; Oligonucleotides; Pathway interactions; Population; Recovery; Resolution; Retrieval; Site; Specificity; Stem cells; System; Systems Development; Tamoxifen; Time; Tissues; Transplantation; base; cell type; cellular transduction; deep sequencing; design; genetic manipulation; in vivo; non-invasive system; novel; novel strategies; reconstruction; tissue reconstruction; virtual

ABSTRACT A complete understanding of immune system development and homeostasis would require the mapping of every immune cell's origin and eventual fate. A powerful approach for high-resolution cell fate mapping involves “DNA barcodes”, i.e. unique short sequences of DNA that are inserted into the genome of cells under study. One drawback of this approach is the necessity of cell isolation and transplantation, which limits it to only a few cell types, destroys the native tissue environment and introduces biases due to transplantation. We propose to develop an approach that introduces DNA barcodes in vivo by genetic manipulation, i.e. without cell isolation or transplantation. Crucially, DNA barcodes would be introduced in a time-controlled and cell type-specific manner, allowing the reconstruction of developmental kinetics and precursor-product relationships in the immune system. In Aim 1, we will validate and optimize the inducible DNA barcoding system. In Aim 2, we will use this system to barcode dendritic cell progenitors and analyze their clonal contribution to mature dendritic cell subsets. These studies would establish a novel experimental system for clonal cell analysis in the immune system, and apply it to the development of a critical immune cell type in unperturbed animals.

抽象的 对免疫系统发育和体内平衡的完整理解需要 绘制每个免疫细胞的起源和最终命运的高分辨率细胞的强大方法。 命运图谱涉及“DNA条形码”，即插入到DNA中的独特的短DNA序列。 这种方法的一个缺点是需要细胞分离和分析。 移植仅限于几种细胞类型，破坏了天然组织环境， 我们建议开发一种引入 DNA 的方法。 通过基因操作在体内形成条形码，即无需细胞分离或移植，最重要的是，DNA。 条形码将以时间控制和细胞类型特定的方式引入，从而允许 重建免疫系统中的发育动力学和前体-产物关系。 在目标 1 中，我们将验证和优化诱导型 DNA 条形码系统。在目标 2 中，我们将使用该系统。 系统对树突状细胞祖细胞进行条形码分析并分析其对成熟树突状细胞的克隆贡献 这些研究将为克隆细胞分析建立一个新的实验系统。 免疫系统，并将其应用于未受干扰的动物中关键免疫细胞类型的发育。