In the literature, there are few high-throughput screens or even methods for high-throughput screens of recombinant adeno-associated virus (rAAV) production despite potential benefits to research and production. In this study, a generalizable high-throughput relative rAAV titration method is examined within the context of an siRNA screen as siRNA knockdown is a common means of pathway engineering in bioproduction. Crude samples generated from transfected HEK293T/17 cultures were subjected to quantitative PCR (qPCR) and used to transduce COS7 cells to assess relative differences in genomic and infectious rAAV titer, respectively, at the 384-well scale, evaluating both supernatant and lysed samples. To evaluate relevant differences in titer for conditions that could be used in an actual screen, cultures subjected to an siRNA reverse transfection and subsequent rAAV forward transfection were also tested. The delayed forward rAAV triple-plasmid transfection was not seen to affect the siRNA activity of tested controls, while siRNA transfection was shown to measurably impact rAAV titer. Effective differentiation between infectious titer levels was dependent upon the choice of sample dilution, but trends between qPCR and infectious titer assays were consistent across sample sets.

中文翻译：

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用于高通量筛选的微型 rAAV 生产和滴定

在文献中，尽管对研究和生产有潜在好处，但很少有高通量屏幕甚至重组腺相关病毒 (rAAV) 生产的高通量屏幕方法。在这项研究中，在 siRNA 筛选的背景下检查了一种通用的高通量相对 rAAV 滴定方法，因为 siRNA 敲低是生物生产中通路工程的常用手段。从转染的 HEK293T/17 培养物中产生的粗样品进行定量 PCR (qPCR)，并用于转导 COS7 细胞，以分别在 384 孔规模上评估基因组和感染性 rAAV 滴度的相对差异，评估上清液和裂解样品。为了评估可用于实际筛选的条件的相关滴度差异，还测试了经过 siRNA 反向转染和随后的 rAAV 正向转染的培养物。延迟正向 rAAV 三重质粒转染未见影响测试对照的 siRNA 活性，而 siRNA 转染显示可测量地影响 rAAV 滴度。感染性滴度水平之间的有效区分取决于样本稀释度的选择，但 qPCR 和感染性滴度测定之间的趋势在样本组中是一致的。