Characterization of retroviral infectivity and superinfection resistance during retrovirus-mediated transduction of mammalian cells.
Characterization of retroviral infectivity and superinfection resistance during retrovirus-mediated transduction of mammalian cells.
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逆转录病毒介导的哺乳动物细胞转导过程中逆转录病毒感染性和重复感染抗性的表征
DOI：
10.1038/gt.2017.24
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发表时间：
2017-06
期刊：
影响因子：
5.1
通讯作者：
Wang J
中科院分区：
文献类型：
--
作者：
Liao J;Wei Q;Fan J;Zou Y;Song D;Liu J;Liu F;Ma C;Hu X;Li L;Yu Y;Qu X;Chen L;Yu X;Zhang Z;Zhao C;Zeng Z;Zhang R;Yan S;Wu T;Wu X;Shu Y;Lei J;Li Y;Zhang W;Wang J;Reid RR;Lee MJ;Huang W;Wolf JM;He TC;Wang J
包括慢病毒载体在内的逆转录病毒载体是在哺乳动物细胞中稳定表达转基因或RNA分子的常用工具。它们的用途大致分为两类：转基因和RNA分子的稳定过表达，这需要最大的转导效率；或者使用基于逆转录病毒（RV）的文库进行功能选择，这利用了逆转录病毒双重感染抗性。然而，RV 介导的转导的动态特征尚未得到很好的表征。在这里，我们设计了两种表达双荧光蛋白和抗生素标记的基于鼠干细胞病毒的逆转录病毒载体，并分析了病毒颗粒的生产效率和病毒颗粒的稳定性、不同细胞类型的动态感染性和重复感染抗性，以及提高转导效率的策略。我们发现转染后 60 至 72 小时内病毒粒子产量最高。储存3天后，收集的病毒颗粒上清液的稳定性下降>60%。我们发现，RV 感染性在测试的人类癌细胞系中存在巨大差异，而低转导效率则通过病毒滴度增加、感染持续时间延长和/或重复感染而部分克服。此外，我们证明了 RV 受体 PIT1 和 PIT2 在分析的细胞中低表达，并且 PIT1 和/或 PIT2 过表达显着提高了某些细胞系中的转导效率。因此，我们的研究结果为逆转录病毒介导的基因传递的最佳条件提供了丰富的信息。
Retroviral vectors including lentiviral vectors are commonly used tools to stably express transgenes or RNA molecules in mammalian cells. Their utilities are roughly divided into two categories, stable overexpression of transgenes and RNA molecules, which requires maximal transduction efficiency, or functional selection with retrovirus (RV)-based libraries, which takes advantage of retroviral superinfection resistance. However, the dynamic features of RV-mediated transduction are not well characterized. Here, we engineered two murine stem cell virus-based retroviral vectors expressing dual fluorescence proteins and antibiotic markers, and analyzed virion production efficiency and virion stability, dynamic infectivity and superinfection resistance in different cell types, and strategies to improve transduction efficiency. We found that the highest virion production occurred between 60 and 72 h after transfection. The stability of the collected virion supernatant decreased by> 60% after 3 days in storage. We found that RV infectivity varied drastically in the tested human cancer lines, while low transduction efficiency was partially overcome with increased virus titer, prolonged infection duration and/or repeated infections. Furthermore, we demonstrated that RV receptors PIT1 and PIT2 were lowly expressed in the analyzed cells, and that PIT1 and/or PIT2 overexpression significantly improved transduction efficiency in certain cell lines. Thus, our findings provide resourceful information for the optimal conditions of retroviral-mediated gene delivery.
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