Endogenous microRNA clusters outperform chimeric sequence clusters in Chinese hamster ovary cells.
Bottom Line: 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.See accompanying commentary by Baik and Lee DOI: 10.1002/biot.201300503.
Affiliation: Department of Biotechnology, Boku University Vienna, Austria; ACIB GmbH, Austrian Centre of Industrial Biotechnology, Graz, Austria.Show MeSH
Mentions: The chimeric miR-15b/16-2 and miR-221/222 clusters were created by concatenation of miRNA expression plasmids with artificial miRNA constructs (Fig. 1) as previously described [26, 32]. In short, the chimeric miRNAs, consisting of the mature CHO miRNA sequences with restriction sites on either end, and an optimized murine loop sequence (Supporting information, Table 1), were cloned into the 3' untranslated region (3'UTR) of emerald green fluorescent protein (emGFP) located in the pcDNA6.2–GW/EmGFP–miR vector (BLOCK–i™ Pol II miR RNAi Expression Vector Kit, Invitrogen Inc., Carlsbad, CA, USA), already containing artificial flanking regions. One of the two corresponding chimeric cluster miRNAs was cut out, including the artificial flanking regions, and inserted into the plasmid with the other chimeric cluster miRNA for artificial cluster generation (Fig. 1) according to the manufacturer's instructions.
Affiliation: Department of Biotechnology, Boku University Vienna, Austria; ACIB GmbH, Austrian Centre of Industrial Biotechnology, Graz, Austria.