Abstract
MicroRNAs (miRNAs) are small non-coding RNAs (∼22 nucleotides) which regulate gene expression by silencing mRNA translation. MiRNAs are transcribed as long primary transcripts, which are enzymatically processed by Drosha/Dgcr8, in the nucleus, and by Dicer in the cytoplasm, into mature miRNAs. The importance of miRNAs for coordinated gene expression is commonly accepted. Consequentially, there is a growing interest in the application of miRNAs to improve phenotypes of mammalian cell factories such as Chinese hamster ovary (CHO) cells. Few studies have reported the targeted over-expression of miRNAs in CHO cells using vector-based systems. These approaches were hampered by limited sequence availability, and required the design of “chimeric” miRNA genes, consisting of the mature CHO miRNA sequence encompassed by murine flanking and loop sequences. Here we show that the substitution of chimeric sequences with CHO-specific sequences for expression of miRNA clusters yields significantly higher expression levels of the mature miRNA in the case of miR–221/222 and miR–15b/16. Our data suggest that the Drosha/Dgcr8-mediated excision from primary transcripts is reduced for chimeric miRNA sequences compared to the endogenous sequence. Overall, this study provides important guidelines for the targeted over-expression of clustered miRNAs in CHO cells.See accompanying commentary by Baik and Lee DOI: 10.1002/biot.201300503
Highlights
The advantage of Chinese hamster ovary (CHO) cells over microbial production systems is that they can produce proteins with human-like post translational modifications [1]
An empty vector was used as negative control that consisted of the same expression cassette with cytomegalovirus (CMV) promoter, emerald green fluorescent protein (emGFP), but no miRNA insert in the emGFP 3’ untranslated region (3’UTR)
Each construct was transfected into a recombinant CHO cell line producing an Epo-Fc fusion protein in three independent replicates
Summary
The advantage of Chinese hamster ovary (CHO) cells over microbial production systems is that they can produce proteins with human-like post translational modifications [1]. MicroRNAs (miRNAs) are increasingly considered as promising tools for CHO cell line development as they were shown to be essential regulators of cellular functions that support cell cycle progression and protein expression (for example [18,19,20])
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