Titering lentiviral vectors: comparison of DNA, RNA and marker expression methods
Titering lentiviral vectors: comparison of DNA, RNA and marker expression methods
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DOI：
10.1038/sj.gt.3301731
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发表时间：
2002-09-01
期刊：
影响因子：
5.1
通讯作者：
Cornetta, K
中科院分区：
文献类型：
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作者：
Sastry, L;Johnson, T;Cornetta, K
为了更好地表征慢病毒载体上清液，我们比较了三种滴度评估方法。这些滴度方法包括评估上清液中的载体RNA序列、转导细胞中的DNA序列以及转导细胞中的载体表达(使用表达绿色荧光蛋白的载体GFP)。为了分析RNA和DNA，我们建立了一种实时检测慢病毒包装序列的方法，并使用该方法对载体序列的数量进行了定量。用流式细胞仪检测GFP的表达。由于功能滴度(DNA和GFP表达滴度)取决于转导效率，我们计算了慢病毒载体RRL-CMV-GFP在293、HeLa或Mus Dunni细胞转导后的滴度。在转导后4天和14天提取基因组DNA，并以质粒标准测定载体DNA分子的数量。在三种细胞系中，293细胞的转导效率最高(PCR估计RRL-CMV-GFP载体的DNA滴度在第14天为2.52+/-0.25×10(6)分子/毫升，在第4天为2.31+/-0.15×10(6)分子/毫升)。根据GFP表达计算滴度时，293细胞也获得了最高滴度(第14天为0.26+/-0.04×10(6)TU/ml，第4天为0.24+/-0.03 10(6)TU/ml)。通过GFP表达试验获得的滴度比DNA分析获得的滴度低大约一个对数，这表明载体表达的可变性可能低估了滴度。直接从载体上清液中获得的RNA滴度相对于一个质粒标准表明，RNA滴度显著高于DNA(类似于10(3)倍)和GFP滴度(类似于10(4)倍)。为了证明慢病毒探针和引物可以用于多种慢病毒载体的滴定，我们还用实时荧光定量聚合酶链式反应方法测定了另外两个HIV1衍生载体RRL-PGK-GFP(6.1+/-1.4×10(5)分子/毫升)和SMPU-RRE-BN(1.26+/-0.2×10(6)分子/毫升)的DNA滴度。我们的结论是，在测试的三种方法中，通过转导细胞的DNA分析评估效价提供了最可靠的功能效价估计，因为这些方法最不可能受到缺陷干扰颗粒和载体表达水平等因素的影响。所描述的实时荧光聚合酶链式反应方法为慢病毒滴定提供了一种可重复性的方法，并且可以应用于各种载体，而与转基因无关。
To better characterize lentiviral vector supernatants, we compared three methods of titer assessment. These titer methods include assessment of vector RNA sequences in supernatants, DNA sequences in transduced cells, and vector expression in transduced cells (using a vector which expressed the green fluorescence protein, GFP). For analysis of RNA and DNA, we developed a real-time PCR method for detecting the lentiviral packaging sequence and used this methodology to quantitate the number of vector sequences. Vector expression was assessed by flow cytometric analysis for GFP. As functional titers (DNA and GFP expression titers) are dependent on transduction efficiency, we calculated the titer of a lentiviral vector, RRL-CMV-GFP, after transduction of 293, HeLa, or Mus dunni cells. Genomic DNA was extracted at 4 and 14 days after transduction and the number of vector DNA molecules was determined against a plasmid standard. Of the three cell lines tested, 293 cells provided the highest rate of transduction (PCR estimated DNA titer for RRL-CMV-GFP vector was 2.52 +/- 0.25 x 10(6) molecules/ml at 14 days, and 2.31 +/- 0.15 x 10(6) molecules/ml at 4 days). When titer was calculated based on GFP expression, the highest titer was also obtained on 293 cells (0.26 +/- 0.04 x 10(6) TU/ml at 14 days, and 0.24 +/- 0.03 10(6) TU/ml at 4 days). The titers obtained by GFP expression assay were approximately one log lower than those obtained by DNA analysis suggesting that variability in vector expression may underestimate titer Measurement of RNA titers directly from vector supernatants against a plasmid standard indicated that the RNA titers are substantially higher than the DNA (similar to10(3)-fold) and GFP titers (similar to10(4)-fold). To show that the lentiviral probe and primers could be used for titering a variety of lentiviral vectors, we have also used the real-time PCR method to determine the DNA titers of two other HIV1 derived vectors, RRL-PGK-GFP (6.1 +/- 1.4 x 10(5) molecules/ml), and SMPU-RRE-BN (1.26 +/- 0.2 x 10(6) molecules/ml). We conclude that of the three methods tested, titers assessed by DNA analysis of transduced cells provide the most reliable estimate of functional titers as these are least likely to be influenced by factors, such as defective interfering particles and vector expression levels. The real-time PCR method described offers a reproducible method for lentiviral titering and can be applied to a wide variety of vectors, regardless of transgene.