Louisiana State University Health Sciences Center, New Orleans, 70112, USA.
J Virol Methods. 2009 May;157(2):113-21. doi: 10.1016/j.jviromet.2008.11.021. Epub 2009 Jan 20.
During the past 12 years, lentiviral vectors have emerged as valuable tools for transgene delivery because of their ability to transduce nondividing cells and their capacity to sustain long-term transgene expression. Despite significant progress, the production of high-titer high-quality lentiviral vectors is cumbersome and costly. The most commonly used method to produce lentiviral vectors involves transient transfection using calcium phosphate (CaP)-mediated precipitation of plasmid DNAs. However, inconsistencies in pH can cause significant batch-to-batch variations in lentiviral vector titers, making this method unreliable. This study describes optimized protocols for lentiviral vector production based on polyethylenimine (PEI)-mediated transfection, resulting in more consistent lentiviral vector stocks. To achieve this goal, simple production methods for high-titer lentiviral vector production involving transfection of HEK 293T cells immediately after plating were developed. Importantly, high titers were obtained with cell culture media lacking serum or other protein additives altogether. As a consequence, large-scale lentiviral vector stocks can now be generated with fewer batch-to-batch variations and at reduced costs and with less labor compared to the standard protocols.
在过去的 12 年中,慢病毒载体因其能够转导非分裂细胞和维持长期转基因表达的能力而成为转基因传递的有价值的工具。尽管取得了重大进展,但高滴度、高质量慢病毒载体的生产仍然繁琐且昂贵。生产慢病毒载体最常用的方法是使用磷酸钙 (CaP) 介导的质粒 DNA 沉淀进行瞬时转染。然而,pH 值的不一致会导致慢病毒载体滴度在批次间产生显著变化,从而使这种方法不可靠。本研究描述了基于聚乙二亚胺 (PEI) 介导转染的优化慢病毒载体生产方案,从而产生更一致的慢病毒载体库存。为了实现这一目标,开发了简单的生产方法,可在 HEK 293T 细胞铺板后立即进行高滴度慢病毒载体的转染。重要的是,完全不含血清或其他蛋白添加剂的细胞培养基也可获得高滴度。因此,与标准方案相比,现在可以使用更少的批次间变化、更低的成本和更少的劳动力来生成大规模的慢病毒载体库存。