Production of Adeno-Associated Virus Vector Serotype rh.10 and Optimization of Its Purification via Chloroform Extraction.

IF 1.7 4区 医学 Q3 PHARMACOLOGY & PHARMACY
Naoki Tamura, Kanzo Suzuki, Hirono Shiraki, Issei Waguri, Eri Segi-Nishida
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Abstract

Recombinant adeno-associated virus (AAV) vectors are widely used for manipulating gene expression. AAVrh.10 is a highly infectious AAV serotype for the central nervous system and various tissues. Owing to its potential use in research, we aimed to optimize the production strategy and develop a simple purification protocol for the AAVrh.10 vector. In this study, we explored a simple production and purification strategy for the AAVrh.10 vector via chloroform extraction and ultrafiltration. Initially, we evaluated the optimal conditions for AAVrh.10-CAG-GFP production using AAV-293 cells. AAVrh.10-CAG-GFP was successfully produced in a serum-free medium after plasmid transfection. Moreover, the culture medium contained a substantial amount of the virus. Therefore, both AAVrh.10-containing cell lysate and culture medium should be used to prepare the AAVrh.10 viral vector. To purify and concentrate AAVrh.10-CAG-GFP from the crude lysate and medium, we optimized the chloroform extraction and ultrafiltration strategies. Subsequently, purified AAVrh.10-CAG-GFP was used to infect HEK-293T cells. Overall, this study provides a simple and effective AAVrh.10 vector preparation strategy for basic and preclinical research.

rh血清型腺相关病毒载体的制备。10氯仿萃取纯化工艺的优化。
重组腺相关病毒(AAV)载体被广泛用于操纵基因表达。AAVrh。10型是一种对中枢神经系统和各种组织具有高度传染性的AAV血清型。由于其在研究中的潜在用途,我们旨在优化生产策略并开发一种简单的AAVrh纯化方案。10向量。在这项研究中,我们探索了一种简单的AAVrh的生产和纯化策略。10载体经氯仿提取和超滤。首先,我们评估了AAVrh的最佳条件。利用AAV-293细胞生产10-CAG-GFP。AAVrh。质粒转染后,在无血清培养基中成功生成10-CAG-GFP。此外,培养基中含有大量的病毒。因此,两者都是。使用含10的细胞裂解液和培养基制备AAVrh。10病毒载体。提纯:提纯和浓缩AAVrh从粗裂解液和培养基中提取10-CAG-GFP,优化了氯仿提取和超滤策略。随后,纯化AAVrh。10-CAG-GFP感染HEK-293T细胞。总的来说,本研究提供了一种简单有效的AAVrh。基础和临床前研究的载体制备策略。
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来源期刊
CiteScore
3.50
自引率
5.00%
发文量
247
审稿时长
2 months
期刊介绍: Biological and Pharmaceutical Bulletin (Biol. Pharm. Bull.) began publication in 1978 as the Journal of Pharmacobio-Dynamics. It covers various biological topics in the pharmaceutical and health sciences. A fourth Society journal, the Journal of Health Science, was merged with Biol. Pharm. Bull. in 2012. The main aim of the Society’s journals is to advance the pharmaceutical sciences with research reports, information exchange, and high-quality discussion. The average review time for articles submitted to the journals is around one month for first decision. The complete texts of all of the Society’s journals can be freely accessed through J-STAGE. The Society’s editorial committee hopes that the content of its journals will be useful to your research, and also invites you to submit your own work to the journals.
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