An improved method for generating retroviral producer clones for vectors lacking a selectable marker gene
An improved method for generating retroviral producer clones for vectors lacking a selectable marker gene
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DOI：
10.1006/bcmd.1998.0184
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发表时间：
1998-05-15
影响因子：
2.3
通讯作者：
Vanin, EF
中科院分区：
文献类型：
--
作者：
Persons, DA;Mehaffey, MG;Vanin, EF
大多数用于临床前和临床研究的逆转录病毒载体都含有一个可选择的标记基因，以促进产生生产者克隆，然而，这种基因在靶细胞中的表达往往是不受欢迎的，因为这可能改变细胞表型并引发宿主免疫反应。不幸的是，对于缺乏可选择标记基因的载体，高效识别高滴度的生产者克隆仍然是一个问题，缺乏标准的方法学。尽管最近在筛选技术方面有所改进，以便在不使用可选择标记的情况下识别高滴度生产者克隆，但解决方案是。在起始细胞群体中出现高滴度克隆的频率非常低的根本问题还没有出现。我们已经开发出一种策略，在要筛选的转导细胞群体中，大大增加产生病毒的克隆的频率，包括那些具有高滴度的克隆。这种方法依赖于通过逆转录病毒包装和载体质粒的共转染在293T细胞中产生的高滴度载体制剂的使用，缺乏可选择标记的载体的病毒制剂被用于在高复数感染(MOT)下重复转导指数增长的包装细胞，所产生的多克隆产生细胞群中的每个细胞都包含未重排载体基因组的多个副本。通过对条件培养液中病毒RNA的狭缝印迹分析，超过95%的来自该群体的克隆产生了载体颗粒。鉴定出许多克隆，估计效价在10(5)-10(6)之间。这些效价是用标准的载体基因组传播试验确认的。这种方法显著提高了在不进行大规模筛查的情况下，轻松识别缺乏可选择标记的高滴度克隆的能力，并应有助于简化基因标记和治疗载体的常规使用。(C)1998年学术出版社。
Most retroviral vectors used in preclinical and clinical studies contain a selectable marker gene to facilitate the generation of producer clones, However, the expression of such genes in target cells is often undesirable since this may modify cellular phenotype and invoke a host immune response, Unfortunately, the efficient identification of high-titer producer clones for vectors lacking a selectable marker gene continues to be problematic and lacking for a standard methodology. Despite recent improvements in the screening techniques for identifying high-titer producer clones without the aid of a selectable marker, a solution to. the fundamental problem of the very low frequency occurrence of high-titer clones within the starting cell population has not emerged. We have developed a strategy which greatly increases the frequency of virus-producing clones, including those with high-titer, within the population of transduced cells to be screened. This approach relies on the use of high-titer vector preparations generated in 293T cells by co-transfection of retroviral packaging and vector plasmids, Viral preparations of a vector lacking a selectable marker were used to repeatedly transduce exponentially growing packaging cells at a high multiplicity of infection (MOT), Each cell in the resulting polyclonal population of producer cells contained multiple copies of the unrearranged vector genome. Greater than 95% of the clones derived from this population produced vector particles as judged by slot blot analysis of viral RNA from conditioned media. Numerous clones with estimated titers of 10(5)-10(6) were identified. These titers were confirmed using a standard vector genome transmission assay. This approach significantly enhances the ability, without large scale screening, to easily identify high-titer clones lacking a selectable marker and should facilitate the routine use of simplifed gene marking and therapeutic vectors. (C) 1998 Academic Press.