Optimizing MERFISH to allow multiplexed measurement of developmental and tonotopicgene expression gradients in the cochlea

优化 MERFISH 以允许对耳蜗中的发育和音调基因表达梯度进行多重测量

• 批准号: 10653753
• 负责人: Andrew K Groves
• 金额: $ 24万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10653753
• 关键词: Adult; Age; Aging; Ammonium Sulfate; Apical; Basilar Membrane; Brain; Buffers; Cell Separation; Cell Size; Cells; Cellular Morphology; Cochlea; Communities; Cryoultramicrotomy; Data; Decalcification; Development; Developmental Gene; Electrophysiology (science); Equilibrium; Fluorescent in Situ Hybridization; Frequencies; Gene Expression; Gene Structure; Genes; Goals; Hair Cells; Hearing; Histologic; Imaging Techniques; Injury; Length; Measurement; Measures; Mechanics; Medicine; Messenger RNA; Methods; Molecular; Mus; Neonatal; Neurons; Organ; Organ of Corti; Pharmaceutical Preparations; Physiologic Ossification; Physiology; Preparation; Property; Protein Isoforms; Proteins; Protocols documentation; RNA Splicing; Research Personnel; Resolution; Retina; Sensory; Site; Structure; Supporting Cell; Techniques; Temporal bone structure; Testing; Tissues; Transcript; age related; base; bone; cell type; cochlear development; college; design; differential expression; experimental study; feasibility testing; instrument; mRNA Transcript Degradation; mineralization; neonatal mice; noise exposure; preservation; single-cell RNA sequencing; sound frequency; spiral ganglion

PROJECT SUMMARY The adult cochlea presents many technical hurdles to researchers studying the normal function of its constituent cell types, and how these cell types respond to injury, aging and degeneration. Because the cochlea is encased in strong bone and contains relatively small numbers of cells compared to other sensory organs such as the retina, isolating cells to characterize their structure, physiology or gene expression is challenging. Moreover, histological methods to make the cochlea more accessible, such as decalcification, are harsh treatments that can degrade mRNA or protein. In this pilot R21 proposal, we will assess the feasibility of a recently-developed technique known as MERFISH – Multiplexed Error-Robust Fluorescence In Situ Hybridization – to simultaneously detect and quantify the expression of hundreds of genes in the cochlea. We believe this technique is sensitive enough to reveal quantitative differences in gene expression along the developmental and tonotopic gradients that exist along the basal-apical axis of the cochlea. Developing a robust protocol to detect, quantify and spatially localize hundreds of genes at once has the ability to transform our understanding of the normal function of the cochlea, and how gene expression changes in the cochlea and spiral ganglion after noise exposure, hair cell loss, aging and drug administration. The goal of this proposal is to demonstrate the feasibility of MERFISH in the neonatal and adult cochlea and to develop protocols to allow MERFISH to be used by the hearing and balance community.

项目概要 成人耳蜗给研究其正常功能的研究人员带来了许多技术障碍 组成细胞类型，以及这些细胞类型如何响应损伤、衰老和退化。 耳蜗被包裹在坚固的骨骼中，与其他感觉器官相比，其细胞数量相对较少 视网膜等器官，分离细胞以表征其结构、生理学或基因表达是 此外，使耳蜗更容易进入的组织学方法（例如脱钙）也具有挑战性。 可能会降解 mRNA 或蛋白质的严厉治疗 在这个试点 R21 提案中，我们将评估以下方案的可行性： 最近开发的技术称为 MERFISH – 多重误差鲁棒原位荧光 杂交——同时检测和量化耳蜗中数百个基因的表达。 相信这项技术足够灵敏，可以揭示基因表达的定量差异 沿耳蜗基尖轴存在的发育梯度和音调梯度。 同时检测、量化和空间定位数百个基因的强大协议具有转化的能力 我们对耳蜗正常功能的理解，以及耳蜗中基因表达的变化 该提案的目标是在噪音暴露、毛细胞损失、衰老和药物管理后的螺旋神经节。 证明 MERFISH 在新生儿和成人耳蜗中的可行性，并制定方案以允许 MERFISH 供听力和平衡界使用。