Figure 2 - Characterization of KO/KD cell lines in CHO-S. A: Out-out PCR of miRNA KO/KD in CHO-S. B: Quantification of mature miRNA by qPCR.
To further validate the miRNA KO/KD cell lines we conducted quantitative real-time PCR of corresponding miRNAs (Figure 2B). The miRNA qPCR results correlate well with the results from out-out PCR, which indicates that miR-128 E7, and miR-181ab E9 are to be considered full miR KO cell lines, but the miR-34c D12 seems to have some miR-34 expression active, despite the negative gDNA PCR result, possibly due to a copy somewhere else in the genome. It should thus only be considered a KD.

Glycoprofiling of miRNA KO/KD cell lines

To functionally characterize the miRNA KO/KD effects, we examined N-glycosylation in the clones. Batch cultures were run in shake flasks, and N-glycosylation profile of secretomes was made from sampling day 4 of the batch culture. Figure 3 shows that miR-128 and miR-34c KO/KD increases the levels of tri- and tetra- antennary structure of N-glycans of the secreted proteins in CHO cells in comparison to the mock cell lines (px458 F6 and px458 H12). On the other hand, miRNA-181 KO cell lines demonstrate no change comparing to mock cell lines. This result indicates that miR-128 and miR-34c may regulate tri- and tetra- antennary levels in N-glycans by either regulating N-acetylglucosamine (GlcNAc) transferases that is responsible for addition of the GlcNAc unit onto β 1,4 (Mgat4a, Mgat4b), and β 1,6 branches (Mgat5), or by regulating UPD-GlcNAc transporters (SLC35A3, SLC35A4, SLC35A5), which is responsible of transporting UPD-GlcNAc from cytosol into Golgi apparatus, as a building block for further GlcNAc transferase reactions.