Finer M H, Dull T J, Qin L, Farson D, Roberts M R
Cell Genesys Inc., Foster City, CA 94404.
Blood. 1994 Jan 1;83(1):43-50.
We describe a novel retroviral packaging system in which high titer amphotropic retrovirus was produced without the need to generate stable producer clones. kat expression vectors, which produce high levels of retroviral vector transcripts and retroviral packaging functions, were transfected into 293 cells followed by virus harvest 48 hours posttransfection. Viral titers as high as 3.8 proviral copies/cell/mL of frozen supernatant in 3T3 cells were obtained, 10- to 50-fold greater than transient viral titers reported using 3T3-based retroviral packaging lines. Cocultivation of primary human CD8+ T lymphocytes after transient transfection of 293 cells with kat plasmids resulted in transduction efficiencies of 10% to 40%, 5- to 10-fold greater compared to cocultivation with a high titer PA317 producer clone and significantly greater than previously reported results for transduction of primary human T lymphocytes with retroviral vectors. Virus produced using the kat system was shown to be free of detectable replication competent retrovirus by an extended provirus mobilization assay, demonstrating that this system is as safe as currently available stable packaging lines. The kat virus production system should be of general use for the rapid production of high titer viral supernatants, as well as for high-efficiency transduction hematopoietic cell types refractory to retroviral transduction.
我们描述了一种新型逆转录病毒包装系统，在该系统中无需生成稳定的生产克隆即可产生高滴度的嗜异性逆转录病毒。将能产生高水平逆转录病毒载体转录本和逆转录病毒包装功能的kat表达载体转染到293细胞中，转染后48小时收获病毒。在3T3细胞中，冷冻上清液的病毒滴度高达3.8个原病毒拷贝/细胞/毫升，比使用基于3T3的逆转录病毒包装细胞系报道的瞬时病毒滴度高10至50倍。用kat质粒瞬时转染293细胞后与原代人CD8 + T淋巴细胞共培养，转导效率为10%至40%，与用高滴度PA317生产克隆共培养相比高5至10倍，且显著高于先前报道的用逆转录病毒载体转导原代人T淋巴细胞的结果。通过延长的原病毒动员试验表明，使用kat系统产生的病毒不含可检测到的具有复制能力的逆转录病毒，这表明该系统与目前可用的稳定包装细胞系一样安全。kat病毒生产系统应普遍用于快速生产高滴度病毒上清液，以及用于高效转导对逆转录病毒转导难治的造血细胞类型。
