Hartigan-O'Connor D, Amalfitano A, Chamberlain J S
Program in Cellular and Molecular Biology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0618, USA.
J Virol. 1999 Sep;73(9):7835-41. doi: 10.1128/JVI.73.9.7835-7841.1999.
Production of gutted, or helper-dependent, adenovirus vectors by current methods is inefficient. Typically, a plasmid form of the gutted genome is transfected with helper viral DNA into 293 cells; the resulting lysate is serially passaged to increase the titer of gutted virions. Inefficient production of gutted virus particles after cotransfection is likely due to suboptimal association of replication factors with the abnormal origins found in these plasmid substrates. To test this hypothesis, we explored whether gutted virus production would be facilitated by transfection into cells expressing various viral replication factors. We observed that C7 cells, coexpressing adenoviral DNA polymerase and preterminal protein, converted plasmid DNA into replicating virus approximately 50 times more efficiently than did 293 cells. This property of C7 cells can be used to greatly increase the efficiency of gutted virus production after cotransfection of gutted and helper viral DNA. These cells should also be useful for generation of recombinant adenovirus from any plasmid-based precursor.
目前通过现有方法生产缺失基因或依赖辅助病毒的腺病毒载体效率较低。通常，将缺失基因的基因组质粒形式与辅助病毒DNA共转染到293细胞中；对所得裂解物进行连续传代以提高缺失基因病毒粒子的滴度。共转染后缺失基因病毒颗粒生产效率低下可能是由于复制因子与这些质粒底物中发现的异常起始位点结合不理想所致。为了验证这一假设，我们探究了将其转染到表达各种病毒复制因子的细胞中是否会促进缺失基因病毒的产生。我们观察到，共表达腺病毒DNA聚合酶和前末端蛋白的C7细胞将质粒DNA转化为复制型病毒的效率比293细胞高约50倍。C7细胞的这一特性可用于在共转染缺失基因和辅助病毒DNA后大幅提高缺失基因病毒的生产效率。这些细胞也应有助于从任何基于质粒的前体生成重组腺病毒。
