title: Breaking limitations of complex culture media: functional non-viral miRNA delivery into pharmaceutical production cell lines.
abstract: MicroRNAs (miRNAs) are promising targets for cell engineering through modulation of crucial cellular pathways. An effective introduction of miRNAs into the cell is a prerequisite to reliably study microRNA function. Previously, non-viral delivery of nucleic acids has been demonstrated to be cell type as well as culture medium dependent. Due to their importance for biopharmaceutical research and manufacturing, Chinese hamster ovary (CHO) and Cevec's Amniocyte Production (CAP) cells were used as host cell lines to investigate transfection reagents with respect to successful delivery of small non-coding RNAs (ncRNAs) and their ability to allow for biological activity of miRNAs and small interfering RNAs (siRNAs) within the cell. In the present study, we screened numerous transfection reagents for their suitability to successfully deliver miRNA mimics into CHO DG44 and CAP cells. Our investigation revealed that the determination of transfection efficiency for a given transfection reagent alone is not sufficient to draw conclusions about its ability to maintain the functionality of the miRNA. We could show that independent from high transfection rates observed for several reagents only one was suitable for efficient introduction of functional miRNA mimics into cells cultured in complex protein production media. We provide evidence for the functionality of transferred ncRNAs by demonstrating siRNA-mediated changes in protein levels and cellular phenotype as well as decreased twinfilin-1 (twf-1) transcript levels by its upstream miR-1 regulator. Furthermore, the process could be shown to be scalable which has important implications for biotechnological applications.
authors.affiliation: Institute of Applied Biotechnology, University of Applied Sciences Biberach, Hubertus-Liebrecht-Strasse 35, 88400 Biberach, Germany; University of Ulm, Faculty of Medicine, Albert-Einstein-Allee 11, 89081 Ulm, Germany. Electronic address: simon.fischer@hochschule-bc.de.
authors.forename: Simon
authors.initials: S
authors.lastname: Fischer
authors.forename: Andreas
authors.initials: A
authors.lastname: Wagner
authors.forename: Aron
authors.initials: A
authors.lastname: Kos
authors.forename: Armaz
authors.initials: A
authors.lastname: Aschrafi
authors.forename: René
authors.initials: R
authors.lastname: Handrick
authors.forename: Juergen
authors.initials: J
authors.lastname: Hannemann
authors.forename: Kerstin
authors.initials: K
authors.lastname: Otte
mesh_terms: D000818:Animals; D016466:CHO Cells; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D003470:Culture Media / Q000737:chemistry / Q000378:metabolism; D018014:Gene Transfer Techniques; D006801:Humans; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D008840:Microfilament Proteins / Q000096:biosynthesis / Q000235:genetics; D004364:Pharmaceutical Preparations; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics; D022661:RNA, Untranslated / Q000737:chemistry / Q000235:genetics*
chemical_list: D003470:Culture Media; D035683:MicroRNAs; D008840:Microfilament Proteins; D004364:Pharmaceutical Preparations; D034741:RNA, Small Interfering; D022661:RNA, Untranslated; C485188:TWF1 protein, human; D011505:Protein-Tyrosine Kinases
keywords: CAP; CHO; ScreenFect A; Transfection; microRNA