Simplified production and concentration of lentiviral vectors to achieve high transduction in primary human T cells.
Simplified production and concentration of lentiviral vectors to achieve high transduction in primary human T cells.
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DOI：
10.1186/1472-6750-13-98
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发表时间：
2013-11-12
影响因子：
3.5
通讯作者：
Brennan FM
中科院分区：
文献类型：
--
作者：
Cribbs AP;Kennedy A;Gregory B;Brennan FM
慢病毒载体已成为在分裂和非分裂细胞中传递转基因的有效载体。许多不同的载体设计修改提高了生物安全性和转基因表达。然而，尽管取得了这些进展，人类原代T细胞的转导仍然具有挑战性，实现有效基因转移的方法通常既昂贵又耗时。在这里，我们提出了一种简单的优化方案，用于慢病毒在原代人CD45RA+ T细胞中的产生和转导。我们表明，高滴度慢病毒的产生与改善原代T细胞转导是依赖于优化的超离心速度在病毒浓度。此外，我们证明了通过对培养条件的简单修改可以提高转导效率。总的来说，当这些修饰结合使用时，在原代人CD45RA+细胞中可实现高达89%的转导效率。本文所描述的优化方案易于实施，并且应该有助于在原代人T细胞中产生具有优越转导效率的高滴度慢病毒，而无需进一步的纯化方法。
Lentiviral vectors have emerged as efficient vehicles for transgene delivery in both dividing and non-dividing cells. A number of different modifications in vector design have increased biosafety and transgene expression. However, despite these advances, the transduction of primary human T cells is still challenging and methods to achieve efficient gene transfer are often expensive and time-consuming. Here we present a simple optimised protocol for the generation and transduction of lentivirus in primary human CD45RA+ T cells. We show that generation of high-titre lentivirus with improved primary T cell transduction is dependent upon optimised ultracentrifuge speed during viral concentration. Moreover, we demonstrate that transduction efficiency can be increased with simple modifications to the culturing conditions. Overall, a transduction efficiency of up to 89% in primary human CD45RA+ cells is achievable when these modifications are used in conjunction. The optimised protocol described here is easy to implement and should facilitate the production of high-titre lentivirus with superior transduction efficiency in primary human T cells without the need for further purification methods.
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影响因子：
5
作者：
Sakoda, T;Kasahara, N;Kedes, L
通讯作者：
Kedes, L
影响因子：
5.1
作者：
Zhang, B;Xia, HQ;Wei, MQ
通讯作者：
Wei, MQ
DOI：
10.1073/pnas.96.6.2988
发表时间：
1999-03-16
影响因子：
11.1
作者：
Case, SS;Price, MA;Crooks, GM
通讯作者：
Crooks, GM
影响因子：
56.9
作者：
Naldini, L;Blomer, U;Trono, D
通讯作者：
Trono, D
影响因子：
5.4
作者：
Dull, T;Zufferey, R;Naldini, L
通讯作者：
Naldini, L