用CRISPR-Cas9试剂生成和分型基因组编辑小鼠的简单方案

Q1 Agricultural and Biological Sciences Current protocols in mouse biology Pub Date : 2020-03-11 DOI:10.1002/cpmo.69

Almudena Fernández, Matías Morín, Diego Muñoz-Santos, Santiago Josa, Andrea Montero, Marcos Rubio-Fernández, Marta Cantero, Julia Fernández, María Jesús del Hierro, Marta Castrillo, Miguel Ángel Moreno-Pelayo, Lluís Montoliu

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引用次数: 10

摘要

本文中描述的简单方案旨在提供所有所需的信息，作为一种全面的，易于遵循的逐步方法，以确保产生预期的基因组编辑小鼠。在这里，我们提供了制备CRISPR-Cas9试剂的方案，用于微注射和电穿孔到单细胞小鼠胚胎中，以创建敲除或敲入小鼠模型，并使用最新的创新下一代测序方法对所产生的后代进行基因分型。©2020 by John Wiley &基本方案1:为您的基因破坏/编辑策略设计最佳RNA指南基本方案2:制备和验证CRISPR-Cas9试剂基本方案3:制备和注射CRISPR-Cas9化合物到受精小鼠卵母细胞基本方案4:基因分型基因组编辑小鼠支持方案:基因分型crispr产生的“indel”突变

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Simple Protocol for Generating and Genotyping Genome-Edited Mice With CRISPR-Cas9 Reagents

The simple protocol described in this article aims to provide all required information, as a comprehensive, easy-to-follow step-by-step method, to ensure the generation of the expected genome-edited mice. Here, we provide protocols for the preparation of CRISPR-Cas9 reagents for microinjection and electroporation into one-cell mouse embryos to create knockout or knock-in mouse models, and for genotyping the resulting offspring with the latest innovative next-generation sequencing methods. © 2020 by John Wiley & Sons, Inc.

Basic Protocol 1: Designing the best RNA guide for your gene disruption/editing strategy

Basic Protocol 2: Preparing and validating CRISPR-Cas9 reagents

Basic Protocol 3: Preparing and injecting CRISPR-Cas9 compounds into fertilized mouse oocytes

Basic Protocol 4: Genotyping genome-edited mice

Support Protocol: Genotyping for CRISPR-generated “indel” mutations

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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.