von Rüden T, Stingl L, Cotten M, Wagner E, Zatloukal K
Research Institute of Molecular Pathology (I.M.P.), Vienna, Austria.
Biotechniques. 1995 Mar;18(3):484-9.
A new procedure is described for the generation of high-titer, helper-free retrovirus vectors employing receptor-mediated, adenovirus-augmented transfection into a standard packaging cell line. Viral titers are increased 30-fold to 100-fold in transiently (> 10(5) infectious units per mL) and stable (> 10(7) infectious units per mL) transfected cells as compared with either CaPO4-mediated transfection or retroviral infection of a packaging cell line. Further, expression of the transduced genes was drastically increased in the transfected cells, but, as expected, there was no difference in transduction efficiency and gene expression in the infected target cells. The increases in viral titers were most likely due to the high number of stable, integrated copies of the vector plasmid DNA in the resulting packaging lines following G418 selection. In addition, experiments generating recombinant retroviruses from non-packaging cell lines are presented. The results suggest that this procedure may be of use to generate high-titer retrovirus vectors in packaging cell lines as well as in primary cells, thus providing a technical basis for in vivo gene transfer upon transplantation of these cells into various organs.
描述了一种新方法，用于生成高滴度、无辅助病毒的逆转录病毒载体，该方法采用受体介导、腺病毒增强转染，将其导入标准包装细胞系。与磷酸钙介导的转染或包装细胞系的逆转录病毒感染相比，在瞬时转染（>10⁵感染单位/毫升）和稳定转染（>10⁷感染单位/毫升）的细胞中，病毒滴度提高了30倍至100倍。此外，转导基因在转染细胞中的表达大幅增加，但正如预期的那样，在感染的靶细胞中，转导效率和基因表达没有差异。病毒滴度的增加很可能是由于在G418选择后，所得包装细胞系中载体质粒DNA有大量稳定整合的拷贝。此外，还展示了从非包装细胞系产生重组逆转录病毒的实验。结果表明，该方法可用于在包装细胞系以及原代细胞中生成高滴度逆转录病毒载体，从而为将这些细胞移植到各种器官中进行体内基因转移提供技术基础。
