Comparison of Lentiviral Packaging Mixes and Producer Cell Lines for RNAi Applications
Comparison of Lentiviral Packaging Mixes and Producer Cell Lines for RNAi Applications
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DOI：
10.1007/s12033-015-9843-8
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发表时间：
2015-06-01
影响因子：
2.6
通讯作者：
Nuernberger, Sylvia
中科院分区：
文献类型：
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作者：
Albrecht, Christian;Hosiner, Stefanie;Nuernberger, Sylvia
慢病毒转导是用于RNA干扰应用的高效DNA递送方法。然而，获得shRNA和miRNA编码载体的高慢病毒滴度是具有挑战性的，因为已经显示shRNA和miRNA盒降低慢病毒滴度。在这项研究中，我们比较了四种市售包装混合物和两种生产细胞系，以优化基因沉默实验的慢病毒生产。将编码miRNA序列和翠绿色绿色荧光蛋白的慢病毒载体与ViraPower（TM）、Lenti-X（TM）HTX、MISSIONA（R）慢病毒或反式慢病毒（TM）包装混合物在HEK-293 T或293 FT细胞中共转染。用含病毒的上清液转导HeLa细胞后，通过流式细胞仪测定慢病毒滴度。在这两种细胞系中，使用MISSIONA（R）慢病毒包装混合物获得的慢病毒滴度最高，其次是ViraPower（TM）、Lenti-X（TM）HTX和Trans-Lentiviral（TM）。平均而言，HEK-293 T细胞产生的慢病毒滴度比293 FT细胞高6.2倍（p < 0.001）。使用MISSIONA（R）慢病毒包装混合物和HEK-293 T细胞的组合，与其他包装混合物和生产细胞系相比，慢病毒滴度高出48.5倍。在这项工作中描述的包装混合物和细胞系的优化选择应有助于生产高滴度的慢病毒用于基因沉默实验。
Lentiviral transduction is a highly efficient DNA delivery method for RNA interference applications. However, obtaining high lentiviral titers of shRNA and miRNA encoding vectors is challenging, since shRNA and miRNA cassettes have been shown to reduce lentiviral titers. In this study, we compare four commercially available packaging mixes and two producer cell lines in order to optimize lentiviral production for gene silencing experiments. Lentiviral vectors encoding a miRNA sequence and emerald green fluorescence protein were co-transfected with ViraPower (TM), Lenti-X (TM) HTX, MISSIONA (R) Lentiviral or Trans-Lentiviral (TM) packaging mix in HEK-293T or 293FT cells. After transducing HeLa cells with virus-containing supernatant, lentiviral titers were determined by flow cytomerty. In both cell lines, the highest lentiviral titer was obtained with MISSIONA (R) Lentiviral packaging mix, followed by ViraPower (TM), Lenti-X (TM) HTX, and Trans-Lentiviral (TM). On average, HEK-293T cells produced 6.2-fold higher lentiviral titers than 293FT cells (p < 0.001). With the combination of MISSIONA (R) Lentiviral packaging mix and HEK-293T cells, an up to 48.5-fold higher lentiviral titer was reached compared to other packaging mixes and producer cell lines. The optimized selection of packaging mix and cell line described in this work should facilitate the production of high-titer lentiviruses for gene silencing experiments.