Quantification of lentiviral vector copy numbers in individual hematopoietic colony-forming cells shows vector dose-dependent effects on the frequency and level of transduction
Quantification of lentiviral vector copy numbers in individual hematopoietic colony-forming cells shows vector dose-dependent effects on the frequency and level of transduction
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DOI：
10.1038/gt.2010.163
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发表时间：
2011-05-01
期刊：
影响因子：
5.1
通讯作者：
Galy, A.
中科院分区：
文献类型：
--
作者：
Charrier, S.;Ferrand, M.;Galy, A.
慢病毒载体是基因转移的有效工具，可将不同数量的原病毒 DNA 拷贝整合到不同比例的细胞中。因此，细胞群体的转导水平可能取决于影响该群体中载体整合事件的频率和/或分布的实验参数。此类分析需要测量单个细胞中的载体拷贝数（VCN）。为了在单细胞水平评估造血祖细胞的转导，我们使用适应的定量 PCR (Q-PCR) 方法测量了单个集落形成细胞 (CFC) 单位中的 VCN。使用携带已知数量的载体整合的特征细胞系测试了该方法的可行性、再现性和敏感性。通过将 CFC 中的数据与基因表达或计算值相关联来验证该方法，并发现该方法稍微低估了 VCN。尽管如此，这种 CFC 上的 Q-PCR 对于比较不同感染方案和不同载体的转导水平很有用。增加载体浓度和重复感染是两种不同的策略，它们通过增加转导祖细胞的频率来改善转导。重复感染也增加了整合拷贝的数量，这种影响的程度似乎取决于载体的制备。因此，VCN在造血集落中的分布可能取决于实验条件，包括载体的特征。这应该在离体造血基因治疗研究的背景下仔细评估。基因治疗 (2011) 18, 479-487; doi：10.1038/gt.2010.163； 2010 年 12 月 16 日在线发布
Lentiviral vectors are effective tools for gene transfer and integrate variable numbers of proviral DNA copies in variable proportions of cells. The levels of transduction of a cellular population may therefore depend upon experimental parameters affecting the frequency and/or the distribution of vector integration events in this population. Such analysis would require measuring vector copy numbers (VCN) in individual cells. To evaluate the transduction of hematopoietic progenitor cells at the single-cell level, we measured VCN in individual colony-forming cell (CFC) units, using an adapted quantitative PCR (Q-PCR) method. The feasibility, reproducibility and sensitivity of this approach were tested with characterized cell lines carrying known numbers of vector integration. The method was validated by correlating data in CFC with gene expression or with calculated values, and was found to slightly underestimate VCN. In spite of this, such Q-PCR on CFC was useful to compare transduction levels with different infection protocols and different vectors. Increasing the vector concentration and re-iterating the infection were two different strategies that improved transduction by increasing the frequency of transduced progenitor cells. Repeated infection also augmented the number of integrated copies and the magnitude of this effect seemed to depend on the vector preparation. Thus, the distribution of VCN in hematopoietic colonies may depend upon experimental conditions including features of vectors. This should be carefully evaluated in the context of ex vivo hematopoietic gene therapy studies. Gene Therapy (2011) 18, 479-487; doi:10.1038/gt.2010.163; published online 16 December 2010