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.

摘要 要完全了解免疫系统的发育和稳态， 绘制出每个免疫细胞的起源和最终命运。一种强大的高分辨率细胞 命运映射涉及“DNA条形码”，即插入到DNA中的独特短DNA序列。 正在研究的细胞的基因组。这种方法的一个缺点是必须进行细胞分离， 移植将其限制为仅几种细胞类型，破坏了天然组织环境， 由于移植引入了偏见。我们建议开发一种方法， 通过遗传操作，即在没有细胞分离或移植的情况下，在体内产生条形码。关键是，DNA 条形码将以时间控制和细胞类型特异性的方式引入， 重建免疫系统中的发育动力学和代谢产物-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 对树突状细胞祖细胞进行条形码化并分析其对成熟树突状细胞的克隆贡献的系统 细胞亚群这些研究将建立一个新的实验系统，用于克隆细胞分析， 免疫系统，并将其应用于在未受干扰的动物中开发关键的免疫细胞类型。