Soneoka Y, Cannon P M, Ramsdale E E, Griffiths J C, Romano G, Kingsman S M, Kingsman A J
Department of Biochemistry, University of Oxford, UK.
Nucleic Acids Res. 1995 Feb 25;23(4):628-33. doi: 10.1093/nar/23.4.628.
We have constructed a series of MLV-based retroviral vectors and packaging components expressed from the CMV promoter and carried on plasmids containing SV40 origins of replication. These two features greatly enhanced retroviral gene expression when introduced into cell lines carrying the SV40 large T antigen. The two packaging components, gag-pol and env, were placed on separate plasmids to reduce helper virus formation. Using a highly transfectable human cell line and sodium butyrate to further increase expression of each component, we achieved helper-free viral stocks of approximately 10(7) infectious units/ml by 48 h after transient co-transfection with the three plasmid components. This system can be used both for the generation of high titer retroviral stocks for transduction and for the rapid screening of a large number of MLV gag-pol or env mutants.
我们构建了一系列基于莫洛尼鼠白血病病毒（MLV）的逆转录病毒载体及包装元件，这些元件由巨细胞病毒（CMV）启动子表达，并携带于含有猴病毒40（SV40）复制起点的质粒上。当将这两个元件导入携带SV40大T抗原的细胞系时，它们极大地增强了逆转录病毒基因的表达。两个包装元件，即gag-pol和env，被置于不同的质粒上以减少辅助病毒的形成。使用一种高度可转染的人类细胞系和丁酸钠来进一步增加每个元件的表达，通过与三种质粒元件进行瞬时共转染后48小时，我们获得了滴度约为10⁷感染单位/毫升的无辅助病毒的病毒原液。该系统可用于产生用于转导的高滴度逆转录病毒原液，也可用于快速筛选大量的MLV gag-pol或env突变体。
