Studying immune development at single-cell resolution by DNA barcoding

摘要 要完全了解免疫系统的发育和动态平衡，需要 绘制每个免疫细胞的起源和最终命运的地图。一种实现高分辨率单元的强大方法 命运图涉及“DNA条形码”，即插入到 正在研究的细胞基因组。这种方法的一个缺点是必须进行细胞分离和 仅限于几种细胞类型的移植，破坏了自然组织环境和 引入了由于移植而产生的偏见。我们建议开发一种将DNA引入 通过基因操作体内的条形码，即不需要细胞分离或移植。至关重要的是，DNA 条形码将以时间受控和单元类型特定的方式引入，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DNA条码系统。在《目标2》中，我们将使用这个 对树突状细胞前体细胞进行条形码编码并分析其对成熟树突状细胞克隆贡献的系统 细胞子集。这些研究将建立一种新的克隆细胞分析的实验系统 免疫系统，并将其应用于在无动于衷的动物中发展一种关键的免疫细胞类型。

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.