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

项目摘要 成年人的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应对损伤，衰老和退化。因为 耳蜗被包裹在强壮的骨骼中，与其他感觉器官相比， 分离细胞以表征其结构、生理学或基因表达， 挑战性此外，使耳蜗更容易接近的组织学方法，如脱钙， 可以降解mRNA或蛋白质的苛刻处理。在这项R21试验计划中，我们会评估以下可行性： 最近开发的技术称为MERFISH -多路复用误差鲁棒荧光原位 杂交-同时检测和量化耳蜗中数百个基因的表达。我们 我相信这项技术足够灵敏，可以揭示基因表达的数量差异，沿着 沿着耳蜗的基底-顶端轴沿着存在的发育和音调分布梯度。开发一 一个强大的协议，检测，定量和空间定位数百个基因一次有能力转换 我们对耳蜗正常功能的理解，以及基因表达如何在耳蜗中变化， 噪声暴露、毛细胞丢失、衰老和药物给药后的螺旋神经节。这项提案的目的是 证明MERFISH在新生儿和成人耳蜗中的可行性，并制定方案， MERFISH将由听力和平衡社区使用。

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.