Production of high titre helper-free recombinant retroviral vectors by lipofection.
Production of high titre helper-free recombinant retroviral vectors by lipofection.
复制标题
通过脂转染生产高滴度无辅助重组逆转录病毒载体。
作者：
A. Fassati;Y. Takahara;F. Walsh;G. Dickson
复制缺陷型逆转录病毒载体的高滴度储备液通常通过将克隆的前病毒J DNA稳定引入包装细胞系中来制备，所述包装细胞系反式提供病毒组装所需的所有蛋白质（1）。然而，不同的载体以不同的效率包装，并且转基因的表达和异源内部启动子元件的活性可能变化很大，这取决于对重组前病毒结构的理解不足且通常不明确的特征（2）。因此，通过分离和选择稳定的生产细胞克隆来优化和比较筛选前病毒结构是极其耗时和劳动密集的程序。制备高滴度逆转录病毒原液的一种更快速和更简单的替代方法是通过瞬时转染包装细胞来短期生产病毒。由于标准转染程序（如使用CaPO 4-DNA沉淀物）效率低下，该方法通常产生低病毒滴度（3，4）。然而，最近已经描述了使用CaPO 4-DNA沉淀物和Cos细胞或其他表达SV 40大T抗原的细胞瞬时生产高滴度逆转录病毒原液的方法（5，6）。在这里，我们描述了一种简单的方法，使用高效脂质体介导的基因转移瞬时和可重复地制备重组逆转录病毒载体在滴度超过10 cfu/ml。将亲嗜性包装细胞系Ampli-GPE（7）的培养物接种在60 mm培养皿（1.3 × 106个细胞/cm）中，24小时后，通过暴露于根据制造商（Gibco BRL，佩斯利，苏格兰）制备的Lipofectamine-DNA复合物来转染细胞。
High titre stocks of replication-defective retroviral vectors are generally prepared by stable introduction of cloned proviraJ DNA into packaging cell lines that supply in-trans all the proteins necessary for viral assembly (1). However, different vectors are packaged with different efficiencies and the expression of transgenes and activity of heterologous internal promoter elements may vary widely, dependent upon poorly understood and often ill-defined features of recombinant proviral structure (2). Optimization and comparative screening of proviral structures by isolation and selection of stable producer cell clones is thus an extremely time consuming and labour intensive procedure. A more rapid and simpler alternative for the preparation of high titre retroviral stocks is the short-term production of virus via transient transfection of packaging cells. This approach often yields low viral titres due to the inefficiency of standard transfection procedure such as the use of CaPO4-DNA precipitates (3,4). However, methods have been recently described for the transient production of high titre retroviral stocks using CaPO4-DNA precipitates and Cos cells or other SV40 large T antigen expressing cells (5,6). Here we describe a simple method using high efficiency Lipofectamine-mediated gene transfer to transiently and reproducably prepare recombinant retroviral vectors at titres in excess of 10 cfu/ml. Cultures of the ecotropic packaging cell line Ampli-GPE (7) were seeded in 60 mm dishes (1.3 X 10, cells/cm) and 24 hours later cells were transfected by exposure to Lipofectamine-DNA complexes prepared according to the manufacturer (Gibco BRL, Paisley, Scotland).