Universal Real-Time PCR-Based Assay for Lentiviral Titration
Universal Real-Time PCR-Based Assay for Lentiviral Titration
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DOI：
10.1007/s12033-014-9815-4
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发表时间：
2015-02-01
影响因子：
2.6
通讯作者：
Kulcenty, Katarzyna
中科院分区：
文献类型：
--
作者：
Barczak, Wojciech;Suchorska, Wiktoria;Kulcenty, Katarzyna
慢病毒载体是在分裂和非分裂细胞中稳定基因转移的有效载体。这一特点使慢病毒载体成为分子研究的有力工具。然而，慢病毒在研究和临床试验中的使用需要一种精确和有效的滴定方法。在这项研究中，我们描述了一种基于qPCR的慢病毒载体滴度估计方法(pLV-THM-GFP)。使用WPRE(土拨鼠肝炎病毒转录后调控元件)和白蛋白基因作为模板，建立基于SYBR Green的实时定量聚合酶链式反应方法，可以在整合的慢病毒DNA水平上快速、灵敏、可重复性和准确地评估慢病毒拷贝数。此外，这种优化使得即使在质量非常差和数量很少的材料中也可以测量慢病毒浓度。因此，这种方法为研究人员提供了一种执行低成本评估的工具，其结果高度可重复性。
Lentiviral vectors are efficient vehicles for stable gene transfer in both dividing and non-dividing cells. This feature among others makes lentiviral vectors a powerful tool in molecular research. However, the use of lentiviruses in research studies and clinical trials requires a precise and validated titration method. In this study, we describe a qPCR-based approach for estimation of lentiviral vector titer (pLV-THM-GFP). The use of WPRE (Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element) and albumin genes as templates for an SYBR green-based real-time qPCR method allows for a rapid, sensitive, reproducible, and accurate assessment of lentiviral copy number at an integrated lentiviral DNA level. Furthermore, this optimization enables measurement of lentiviral concentration even in very poor quality and small quantity material. Consequently, this approach provides researchers with a tool to perform low-cost assessment with highly repeatable results.