MiRNA Chaining for Efficient Stable Overexpression to Improve Protein Quantity and Quality in CHO Cells

MicroRNAs (miRNAs), small noncoding RNAs with a length of about 22 nucleotides, harbor the potential to be powerful tools for the genetic engineering of production cell lines like Chinese hamster ovary (CHO) cells. Their ability to regulate multiple targets at once and their potential to fine-tune effect strengths contrast with classical engineering approaches. However, most studies of miRNAs rely on transiently flooding the cells with miRNA mimics. Since this approach is not suitable for long-term cultivation in a bioprocess, stable overexpression of miRNAs becomes more and more important for the biotech industry. Here, the user might be confronted with insufficient overexpression of the miRNA of interest. In this chapter, we present a method for the generation of stable CHO cell lines expressing a miRNA from a plasmid-based system containing multiple copies of the miRNA, allowing tuning of overexpression and regulation.