Austrian Centre of Industrial Biotechnology, Graz, Austria.
University of Natural Resources and Life Sciences, Vienna, Austria.
Sci Rep. 2019 Jun 18;9(1):8689. doi: 10.1038/s41598-019-45159-2.
High-throughput siRNA screens were only recently applied to cell factories to identify novel engineering targets which are able to boost cells towards desired phenotypes. While siRNA libraries exist for model organisms such as mice, no CHO-specific library is publicly available, hindering the application of this technique to CHO cells. The optimization of these cells is of special interest, as they are the main host for the production of therapeutic proteins. Here, we performed a cross-species approach by applying a mouse whole-genome siRNA library to CHO cells, optimized the protocol for suspension cultured cells, as this is the industrial practice for CHO cells, and developed an in silico method to identify functioning siRNAs, which also revealed the limitations of using cross-species libraries. With this method, we were able to identify several genes that, upon knockdown, enhanced the total productivity in the primary screen. A second screen validated two of these genes, Rad21 and Chd4, whose knockdown was tested in additional CHO cell lines, confirming the induced high productivity phenotype, but also demonstrating the cell line/clone specificity of engineering effects.
高通量 siRNA 筛选技术最近才被应用于细胞工厂，以鉴定能够促进细胞向所需表型的新型工程靶点。虽然已经有针对模式生物（如小鼠）的 siRNA 文库，但没有公开的 CHO 特异性文库，这限制了该技术在 CHO 细胞中的应用。这些细胞的优化特别值得关注，因为它们是生产治疗性蛋白的主要宿主。在这里，我们通过将小鼠全基因组 siRNA 文库应用于 CHO 细胞来进行跨物种方法，优化了用于悬浮培养细胞的方案，因为这是 CHO 细胞的工业实践，并且开发了一种用于识别功能 siRNA 的计算方法，该方法还揭示了使用跨物种文库的局限性。使用这种方法，我们能够鉴定出一些基因，这些基因在敲低后，在初步筛选中提高了总生产率。第二个筛选验证了其中两个基因 Rad21 和 Chd4，在其他 CHO 细胞系中测试了它们的敲低，证实了诱导的高生产力表型，但也证明了工程效应的细胞系/克隆特异性。
