title: Endogenous microRNA clusters outperform chimeric sequence clusters in Chinese hamster ovary cells.
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.
authors: Department of Biotechnology, Boku University Vienna, Austria; ACIB GmbH, Austrian Centre of Industrial Biotechnology, Graz, Austria.
authors: Gerald
authors: G
authors: Klanert
authors: Vaibhav
authors: V
authors: Jadhav
authors: Konstantina
authors: K
authors: Chanoumidou
authors: Johannes
authors: J
authors: Grillari
authors: Nicole
authors: N
authors: Borth
authors: Matthias
authors: M
authors: Hackl
mesh_terms: D000818:Animals; D001483:Base Sequence; D001709:Biotechnology; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D005786:Gene Expression Regulation / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D051379:Mice; D035683:MicroRNAs / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010641:Phenotype; D043244:Ribonuclease III / Q000378:metabolism; D014162:Transfection
chemical_list: D035683:MicroRNAs; D043244:Ribonuclease III
keywords: CHO cell; Chimeric sequence; Endogenous miRNA; MiRNA cluster; MicroRNA engineering