Quantitative polymerase chain reaction as a reliable method to determine functional lentiviral titer after ex vivo gene transfer in human mesenchymal stem cells
Quantitative polymerase chain reaction as a reliable method to determine functional lentiviral titer after ex vivo gene transfer in human mesenchymal stem cells
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DOI：
10.1002/jgm.1049
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发表时间：
2007-07-01
影响因子：
3.5
通讯作者：
Schieker, Matthias
中科院分区：
文献类型：
--
作者：
Boecker, Wolfgang;Rossmann, Oliver;Schieker, Matthias
背景人骨髓间充质干细胞（humanmesenchymalstemcells，hMSCs）是体外基因治疗的理想靶细胞，慢病毒是hMSCs基因转移的理想载体，因为慢病毒具有高的转导率和长期的基因表达。然而，由于耗时的细胞选择和病毒滴定，hMSC的衰老可能限制治疗应用。在这里，我们描述了一种快速，可靠的方法来确定功能慢病毒滴度的定量聚合酶链反应（qPCR）后，高效的体外基因转移在hMSCs.Methods慢病毒生产进行了测试与不同类型的包装系统。使用p24 ELISA在细胞培养物上清液中检测剩余的病毒颗粒。通过FACS分析定量慢病毒基因转移效率。结果第三代自失活载体在hMSCs中表现出高效的基因转移。更换4次培养基后，在细胞培养上清液中未检测到病毒抗原，表明4天后不存在感染性颗粒。我们通过qPCR分析观察到病毒稀释度与转基因表达水平之间的线性相关性，因此允许通过定量转基因表达来进行病毒滴度。最后，我们证明，转导的hMSCs保持其干细胞的特性，分化成脂肪，成骨和软骨lineages.Conclusions定量的转基因拷贝数的qPCR是一种快速，可靠的方法来确定功能慢病毒滴度后，离体基因转移在hMSCs。版权所有（C）2007约翰威利父子有限公司
Background Human mesenchymal stem cells (hMSCs) are a promising target for ex vivo gene therapy and lentiviruses are excellent gene transfer vehicles in hMSCs since they achieve high transduction rates with long-term gene expression. Nevertheless, senescence of hMSCs may limit therapeutic applications due to time-consuming cell selection and viral titration. Here, we describe a fast and reliable method to determine functional lentiviral titer by quantitative polymerase chain reaction (qPCR) after highly efficient ex vivo gene transfer in hMSCs.Methods Lentivirus production was tested with different types of packaging systems. Using p24 ELISA remaining viral particles were detected in the cell culture supernatant. The lentiviral gene transfer efficiency was quantified by FACS analysis. Lentiviral titers were determined by qPCR of expressed transgenes.Results Third-generation self-inactivating vectors showed highly efficient gene transfer in hMSCs. No viral antigen was detected in the cell culture supernatant after four media changes, suggesting the absence of infectious particles after 4 days. We observed a linear correlation between virus dilution and level of transgene expression by qPCR analysis, therefore allowing viral titering by quantification of transgene expression. Finally, we demonstrated that transduced hMSCs retained their stem cell character by differentiation towards adipogenic, osteogenic and chondrogenic lineages.Conclusions Quantification of transgene copy numbers by qPCR is a fast and reliable method to determine functional lentiviral titer after ex vivo gene transfer in hMSCs. Copyright (C) 2007 John Wiley & Sons, Ltd.