腺相关病毒的产生、纯化和滴度测定

Q1 Agricultural and Biological Sciences

Current protocols in mouse biology Pub Date : 2018-11-29 DOI:10.1002/cpmo.56

Yong Hong Chen, Megan S. Keiser, Beverly L. Davidson

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引用次数: 16

摘要

腺相关病毒(AAV)载体是研究体内基因功能的典型工具，特别有利于将感兴趣的基因转移到脑组织中。它们作为临床前和临床应用的基因治疗载体显示出巨大的前景。然而，由于病毒本身并不容易获得，因此使用该工具的能力经常受到阻碍。AAV的生产方法有很多。在这里，我们描述了一种基于PEI Max介导的HEK 293细胞三重转染和碘二醇梯度超离心纯化的中小规模(1012-1013个病毒颗粒)生产AAV的简单方法。这些方法将为用户提供足够质量的材料进行体内基因转移。©2018 by John Wiley &儿子,Inc。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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Adeno-Associated Virus Production, Purification, and Titering

Adeno-associated virus (AAV) vectors are exemplary tools for studying gene function in vivo and are particularly favorable for transferring genes of interest into brain tissues. They have shown great promise as a gene therapy vector for preclinical and clinical applications. However, the ability to use this tool is often hampered because the viruses themselves are not readily available. Many methods have been developed for AAV production. Here, we describe a simple method for small- to medium-scale (10¹²-10¹³ viral particles) production of AAV based on Polyethylenimine Max (PEI Max)–mediated triple transfection of HEK 293 cells and purification with iodixanol gradient ultracentrifugation. These methods will provide users with ample material of sufficient quality for performing in vivo gene transfer. © 2018 by John Wiley & Sons, Inc.

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来源期刊

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.