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.