Slide-seq: high-resolution in situ expression profiling for neuropathology

Slide-seq：神经病理学的高分辨率原位表达谱分析

• 批准号: 9349150
• 负责人: Evan Z Macosko
• 金额: $ 267万
• 依托单位: BROAD INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9349150
• 关键词: Adult; Alzheimer' s disease model; Amyloid; Biological; Biological Assay; Biology; Brain; Brain Diseases; Cells; Clinical; DNA; DNA biosynthesis; Development; Disease; Gene Expression; Gene Expression Profiling; Genes; Hippocampus (Brain); Human; In Situ; Laboratories; Measures; Methods; Microscope; Modeling; Molecular; Molecular Computations; Molecular Profiling; Mus; Neurons; Pathologic; Population; Positioning Attribute; Reporting; Resolution; Scheme; Slide; Specimen; Symptoms; Tissues; Transfer RNA; Validation; base; brain tissue; cell type; experimental study; genome-wide; innovation; mouse model; neuropathology; neuropsychiatric disorder; new technology; novel; protein expression; success; tool; transcriptome

ABSTRACT Human brain specimens are one of few available opportunities to directly investigate the molecular underpinnings of neuropsychiatric diseases. Assays of gene and protein expression in situ have revolutionized our understanding of many of these diseases, but despite these advances, for many brain disorders, the tissue changes that give rise to clinical symptoms are poorly understood. Ascertaining the full set of molecular change(s) and the cell type(s) in which each change resides would inform the laboratory models used to study these illnesses, and offer hypotheses for potential new treatments. This proposal outlines a novel technology, Slide-seq, capable of performing whole-transcriptome analysis of intact brain tissue sections at single-cell (~7 μm) resolution. The method applies my recent development of a novel DNA barcoding scheme, based upon split-and-pool DNA synthesis, to construct a microscope slide adorned with a high-diversity bead array capable of recording fine spatial coordinates for each gene, transcriptome-wide. In this proposal, we: (1) describe the molecular, computational, and cell biological innovations necessary to transfer RNA from tissue sections onto this array, and successfully report their spatial positions; (2) outline three core assays for unambiguously measuring the success of each crucial technical step; (3) critically evaluate the single-cell resolution of Slide- seq by profiling the mouse adult hippocampus; and (4) apply Slide-seq to a mouse model of Alzheimer's disease to uncover how surrounding cell populations respond to neuronal amyloid accumulation in the early stages of disease. Together, these experiments will produce a well-validated tool with broad applicability to problems in biology, most especially the analysis of pathological brain tissue.

摘要 人脑标本是为数不多的直接研究分子生物学的机会之一。 神经精神疾病的基础基因和蛋白质表达的原位检测已经发生了革命性的变化 我们对许多这些疾病的理解，但尽管有这些进步，对于许多大脑疾病， 引起临床症状的变化知之甚少。确定全套分子 变化和每种变化所在的细胞类型将告知用于研究的实验室模型 这些疾病，并提供潜在的新治疗方法的假设。该提案概述了一种新技术， Slide-seq，能够在单细胞（~7）水平对完整脑组织切片进行全转录组分析 μm）分辨率。该方法应用了我最近开发的一种新的DNA条形码方案， 分裂和合并DNA合成，以构建装饰有高多样性珠阵列的显微镜载玻片， 记录每个基因（全转录组）的精细空间坐标。在本建议中，我们：（1）描述 分子、计算和细胞生物学创新，将RNA从组织切片转移到 该阵列，并成功地报告了它们的空间位置;（2）概述了三个核心测定， 衡量每个关键技术步骤的成功;（3）严格评估Slide的单细胞分辨率， 通过分析小鼠成年海马体来进行Slide-seq;以及（4）将Slide-seq应用于阿尔茨海默病小鼠模型 揭示周围细胞群如何在早期对神经元淀粉样蛋白积累做出反应 疾病的阶段。总之，这些实验将产生一个经过充分验证的工具，具有广泛的适用性， 生物学中的问题，尤其是病理脑组织的分析。