Transduction efficiency of pantropic retroviral vectors is controlled by the envelope plasmid to vector plasmid ratio
Transduction efficiency of pantropic retroviral vectors is controlled by the envelope plasmid to vector plasmid ratio
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DOI：
10.1021/bp049865x
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发表时间：
2005-01-01
影响因子：
2.9
通讯作者：
Aiyar, A
中科院分区：
文献类型：
--
作者：
Chen, Y;Miller, WM;Aiyar, A
用水泡性口炎病毒包膜G蛋白（VSV-G）假型化的泛嗜性逆转录病毒载体通常通过VSV-G表达质粒的瞬时转染产生，因为VSV-G的组成型表达具有细胞毒性。为了产生泛嗜性载体，将VSV-G表达质粒和载体质粒共转染到包装细胞系，如293-gag-pol中。然而，尚不清楚这一范围是否是生产病媒的最佳范围。本文系统地研究了VSV-G质粒（pVSV-G）与载体质粒的比例对载体生产的影响。为此，通过使用脂质体将pVSV-G和表达增强型绿色荧光蛋白（EGFP）的逆转录病毒载体质粒（pLTR-EGFP）共转染293-gag-pol稳定包装细胞。转染后收集载体并用于转染三种靶细胞系，即3T3成纤维细胞、端粒酶永生化的人二倍体成纤维细胞（HDF）和人肝癌细胞系HuH7。对以不同pVSV-G：pLTR-EGFP比率产生的载体的转导效率进行评估，使得转染到293-gag-pol细胞中的质粒的总量保持恒定。我们的结果表明，当pVSV-G：pLTR-EGFP比率远低于1.0时，转导效率最高。对于3T3和HDF细胞，当pVSV-G：pLTR-EGFP的比例为0.053至0.2时，获得最大的转导效率。在对照培养物中，这种效应的相对大小对于较低的转导效率更大。对于HuH7细胞，有益效果小于使用HDF或3T3细胞时观察到的效果。在不同pVSV-G：pLTR-EGFP比例下产生的载体的转导效率的差异不是由于包装细胞或靶细胞增殖的差异。进一步的表征表明，随着pVSV-G：pLTR-EGFP比例的增加，载体RNA相对于p30（gag）的量减少。这些结果表明，只要表达最低限度足够水平的泛嗜性包膜蛋白，转导效率就随着载体RNA水平的增加而增加。
Pantropic retroviral vectors pseudotyped with vesicular stomatitis virus envelope G protein (VSV-G) are typically produced by transient transfection of the VSV-G expression plasmid because constitutive expression of VSV-G is cytotoxic. To produce pantropic vectors, the VSV-G expression plasmid and the vector plasmid are cotransfected into a packaging cell line, such as 293-gag-pol. Typically, the ratio of VSV-G plasmid to the vector plasmid ranges from 0.33 to 1.0. However, it is not clear that this range is optimal for vector production. In this study we have systematically examined the effect of the ratio of VSV-G plasmid (pVSV-G) to vector plasmid on vector production. For this, 293-gag-pol stable packaging cells were cotransfected with pVSV-G and an enhanced green fluorescent protein- (EGFP-) expressing retroviral vector plasmid (pLTR-EGFP) by use of lipofectamine. Vector was collected following transfection and used to transduce three target cell lines, namely, 3T3 fibroblasts, telomerase-immortalized human diploid fibroblasts (HDF), and the human hepatoma cell line HuH7. Transduction efficiency was evaluated for vectors produced at different pVSV-G:pLTR-EGFP ratios such that the total amount of plasmid transfected into 293-gag-pol cells was kept constant. Our results indicate that transduction efficiency is greatest when the pVSV-G:pLTR-EGFP ratio is substantially below 1.0. For 3T3 and HDF cells, the maximum transduction efficiency was obtained when a ratio of pVSV-G:pLTR-EGFP ranging from 0.053 to 0.2 was used for transfection. The relative magnitude of this effect was greater for lower transduction efficiencies in control cultures. For HuH7 cells, the beneficial effects were smaller than those observed when HDF or 3T3 cells were used. The difference in transduction efficiency for vector produced under various pVSV-G:pLTR-EGFP ratios was not due to differences in the proliferation of packaging cells or target cells. Further characterization showed that the amount of vector RNA relative to p30(gag) decreased as the ratio of pVSV-G:pLTR-EGFP increased. These results indicate that transduction efficiency increases with increasing levels of vector RNA as long as a minimally sufficient level of pantropic envelope protein is expressed.