Optimization of Large-Scale Adeno-Associated Virus (AAV) Production.
Optimization of Large-Scale Adeno-Associated Virus (AAV) Production.
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大规模腺相关病毒 (AAV) 生产的优化。
DOI：
10.1002/cpz1.757
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Blackwood,ErikA
中科院分区：
文献类型：
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作者：
Bilal,AlinaS;Parker,SarahN;Murray,VictoriaB;MacDonnell,LaurenF;Thuerauf,DonnaJ;Glembotski,ChristopherC;Blackwood,ErikA
体内基因操作是从机制上理解疾病和生理过程中基因功能的关键方法。为了促进这一点，在小鼠等流行动物模型中开发了胚胎转基因技术。与更长、更昂贵的转基因方法相比，病毒载体，如腺相关病毒（AAV），由于其相对较低的成本和易于生产而越来越受欢迎，这意味着作为一种生物工具具有更大的通用性。在本文中，我们描述了用于体内有效转导的 AAV 生产和纯化方案。重要的是，我们的方法与其他方法的不同之处在于应用了一种简化的、更具成本效益的方法。通过这种方法，可以在 3 至 4 周内生产多达 2 × 1013 个包含基因组的病毒颗粒 (vp) 或 200 个单位，耗材和试剂的最低成本为 1800 至 2000 美元，每周的人员工作时间少于 15 小时。这里的一个单位定义为 1 × 1011vp，这是我们通过尾静脉注射的每只动物 AAV 的标准剂量。因此，我们的方法提供了 AAV 的生产和纯化，其数量能够转导多达 200 只动物。 © 2023 作者。 Wiley periodicals LLC 发布的当前方案。基本方案 1：AAV 生产基本方案 2：AAV 纯化
Genetic manipulationin vivois a critical method for mechanistically understanding gene function in disease and physiological processes. To facilitate this, embryonic transgenesis in popular animal models like mice has been developed. Compared to the longer, expensive methods of transgenesis, viral vectors, such as adeno‐associated virus (AAV), have grown increasingly in popularity due to their relatively low cost and ease of production, translating to an overall greater versatility as a biological tool. In this article, we describe protocols for AAV production and purification for efficient transductionin vivo. Importantly, our method differs from others in application of a streamlined, more cost‐effective approach. From this method, as many as 2 × 1013genome‐containing viral particles (vp), or 200 units, can be produced within 3 to 4 weeks, with a minimal cost of $1800 to $2000 for supplies and reagents and <15 hr of personnel time per week. A unit here is defined as 1 × 1011vp, our standard dose of AAV per animal, injected via tail vein. Therefore, our method provides production and purification of AAV in quantities capable of transducing up to 200 animals. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: AAV productionBasic Protocol 2: AAV purification