A rapid screening procedure for the identification of high-titer retrovirus packaging clones
A rapid screening procedure for the identification of high-titer retrovirus packaging clones
复制标题
鉴定高滴度逆转录病毒包装克隆的快速筛选程序
作者：
B. Murdoch;Ds Pereira;X. Wu;Je Dick;J. Ellis
我们建立了一种病毒RNA(VRNA)斑点杂交方法，通过包装细胞克隆来快速鉴定高滴度逆转录病毒载体的生产。该程序使用Trizol LS试剂从包装细胞上清液中提纯vRNA，灵敏的斑点印迹分析，以及PhphorImager技术在2天内对包装病毒基因组进行定量。对已知生物滴度的病毒上清液进行的实验表明，vRNA斑点印迹分析非常敏感，斑点强度与病毒滴度直接相关。通常需要分析大约100个产生病毒的细胞克隆，这使得这种方法可以作为快速筛查来识别最高产生病毒的克隆。VRNA斑点印迹试验一致地确定了候选高滴度产生细胞克隆的子集。在三个独立的筛选中，具有最高生物滴度的上清液是由先前定义的候选高滴度生产者克隆产生的。我们的程序极大地促进了病毒滴定：(1)快速消除绝大多数低滴度生产者细胞克隆；(2)准确识别候选高滴度生产者克隆的子集，用于进一步的生物滴定和评估前病毒基因组结构；以及(3)将费力的组织培养操作减少到最低限度。此外，这种方法对分子检测的依赖使其非常适合于分离缺乏药物选择标记的高滴度克隆。
We have developed a viral RNA (vRNA) dot blot assay for rapid identification of high-titer retrovirus vector production by packaging cell clones. The procedure employs Trizol LS reagent to purify vRNA from packaging cell supernatants, a sensitive dot blot assay, and PhosphorImager technology to quantify packaged viral genomes in 2 days. Experiments performed on viral supernatants of known biological titer demonstrated that the vRNA dot blot assay was extremely sensitive and that dot intensity correlated directly with viral titer. It is often necessary to analyze approximately 100 virus producing cell clones, making this method useful as a rapid screen to identify the highest virus producing clones. The vRNA dot blot assay consistently identified a subset of candidate high-titer producer cell clones. In three independent screens the supernatant with the highest biological titer was produced by one of the previously defined candidate high-titer producer clones. Our procedure greatly facilitates virus titration by: (1) rapidly eliminating the vast majority of low-titer producer cell clones; (2) accurately identifying the subset of candidate high-titer producer clones for further biological titration and assessment of the proviral genomic structure; and (3) reducing laborious tissue culture manipulations to a minimum. Furthermore, the reliance of this method on molecular detection makes it ideally suited for the isolation of high-titer clones lacking a drug selection marker.