Whole-Mount In Situ Hybridization in Post-Implantation Staged Mouse Embryos

Q1 Agricultural and Biological Sciences

Current protocols in mouse biology Pub Date : 2020-05-21 DOI:10.1002/cpmo.75

Kristen S. Barratt, Ruth M. Arkell

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引用次数: 5

Abstract

Understanding RNA expression in space and time is a key initial step in dissecting gene function. The ability to visualize gene expression in whole-tissue or whole-specimen preparations, called in situ hybridization (ISH), was first developed 50 years ago. Two decades later, these protocols were adapted to establish robust methods for whole-mount ISH to murine embryos. The precise protocols vary somewhat between early-gestation and mid-gestation mouse embryos; the protocol presented here is optimal for use with post-implantation stage mouse embryos (stages 5.5–9.5 dpc). Routine uses of whole-mount ISH include documenting the wild-type expression pattern of individual genes and comparison of the expression pattern of signature genes (i.e., those that identify particular cells and tissues within an embryo) between wild-type and mutant embryos as part of a phenotyping experiment. This technique remains a mainstay of developmental biology studies and complements the massively parallel assessment of gene expression from dissociated tissues and cells via RNA-sequencing techniques. © 2020 by John Wiley & Sons, Inc.

Basic Protocol 1: Dissection of post-implantation (5.5-9.5 dpc) murine embryos

Basic Protocol 2: Whole-mount in situ hybridization in post-implantation embryos

Basic Protocol 3: Visualization of post-WMISH embryos

Support Protocol 1: Creation of siliconized glass pipettes

Support Protocol 2: Creation of embryo powder

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植入后分期小鼠胚胎的全贴装原位杂交

了解RNA在空间和时间上的表达是解剖基因功能的关键的第一步。在整个组织或整个标本制备中可视化基因表达的能力，称为原位杂交(ISH)，最早是在50年前发展起来的。二十年后，这些协议被改编为建立小鼠胚胎全载ISH的可靠方法。在妊娠早期和妊娠中期的小鼠胚胎中，精确的方案有所不同;本文提出的方案最适合用于植入后阶段的小鼠胚胎(阶段5.5-9.5 dpc)。作为表型实验的一部分，全载ISH的常规用途包括记录单个基因的野生型表达模式和比较野生型和突变胚胎之间的特征基因(即识别胚胎内特定细胞和组织的基因)的表达模式。这项技术仍然是发育生物学研究的支柱，并补充了通过rna测序技术对游离组织和细胞的基因表达进行大规模平行评估。©2020 by John Wiley &基本方案1:植入后(5.5-9.5 dpc)小鼠胚胎的解剖基本方案2:植入后胚胎的全贴装原位杂交基本方案3:wmish后胚胎的可视化支持方案1:创建硅化玻璃移液支持方案2:创建胚胎粉末

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Current protocols in mouse biology Agricultural and Biological Sciences-Animal Science and Zoology

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期刊介绍： Sound and reproducible laboratory methods are the foundation of scientific discovery. Yet, all too often, nuances that are critical for an experiment''s success are not captured in the primary literature but exist only as part of a lab''s oral tradition. The aim of Current Protocols in Mouse Biology is to provide the clearest, most detailed and reliable step-by-step instructions for protocols involving the use of mice in biomedical research. Written by experts in the field and extensively edited to our exacting standards, the protocols include all of the information necessary to complete an experiment in the laboratory—introduction, materials lists with supplier information, detailed step-by-step procedures with helpful annotations, recipes for reagents and solutions, illustrative figures and information-packed tables. Each article also provides invaluable discussions of background information, applications of the methods, important assumptions, key parameters, time considerations, and tips to help avoid common pitfalls and troubleshoot experiments. Furthermore, Current Protocols in Mouse Biology content is thoughtfully organized by topic for optimal usage and to maximize contextual knowledge. Quarterly issues allow Current Protocols to constantly evolve to keep pace with the newest discoveries and developments. Current Protocols in Mouse Biology brings together resources in mouse biology and genetics and provides a mouse protocol resource that covers all aspects of mouse biology. Current Protocols in Mouse Biology also permits optimization of mouse model usage, which is significantly impacted by both cost and ethical constraints. Optimal and standardized mouse protocols ultimately reduce experimental variability and reduce the number of animals used in mouse experiments.