title: Fast and Efficient Transfection of Mouse Embryonic Stem Cells Using Non-Viral Reagents.
abstract: Reliable and efficient DNA and RNA transfection methods are required when studying the role of individual genes in mouse pluripotent stem cells. However, these cells usually grow in tight clusters and are therefore more difficult to transfect than many other cell lines. We have found that transfection is especially challenging when mouse embryonic stem (mES) cells are cultured in the newly described 2i medium, which is based on two chemical inhibitors of differentiation pathways. In the present study we have performed a side-by-side comparison of commercially available, non-viral transfection reagents with regard to their ability to deliver plasmid DNA and siRNA into adherent and/or trypsinized mES cells cultured in 2i medium, assessing transfection rates, plasmid gene expression, siRNA mediated knockdown of Oct4 and viability. Finally, we present a fast and efficient method for transfection of trypsinized mES cells using the liposomal-based Lipofectamine 2000. With only a five-minute long transfection time we obtained at least 85 % transfected cells with 80 % maintained viability. Moreover, this protocol saves up to a day of experimental time since the cells are in suspension at the time of transfection, which allows for immediately re-plating into the appropriate format. This fast, simplified and highly efficient transfection method will be valuable for both basic research and high-throughput applications.
authors.affiliation: Department of Medical Biochemistry and Microbiology, Uppsala University, Box 582, SE-751 23, Uppsala, Sweden.
authors.forename: Christoffer
authors.initials: C
authors.lastname: Tamm
authors.affiliation: Department of Medical Biochemistry and Microbiology, Uppsala University, Box 582, SE-751 23, Uppsala, Sweden.
authors.forename: Sandeep
authors.initials: S
authors.lastname: Kadekar
authors.affiliation: Department of Medical Biochemistry and Microbiology, Uppsala University, Box 582, SE-751 23, Uppsala, Sweden.
authors.forename: Sara
authors.initials: S
authors.lastname: Pijuan-Galitó
authors.affiliation: Department of Medical Biochemistry and Microbiology, Uppsala University, Box 582, SE-751 23, Uppsala, Sweden. cecilia.anneren@ge.com.|GE Healthcare BioSciences AB, Björkgatan 30, Uppsala, SE-751 84, Sweden. cecilia.anneren@ge.com.
authors.forename: Cecilia
authors.identifier: 0000-0002-8447-9543
authors.initials: C
authors.lastname: Annerén
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004247:DNA / Q000235:genetics; D007202:Indicators and Reagents / Q000494:pharmacology*; D008055:Lipids / Q000494:pharmacology; D051379:Mice; D000066450:Mouse Embryonic Stem Cells / Q000187:drug effects*; D010957:Plasmids / Q000235:genetics; D039904:Pluripotent Stem Cells / Q000187:drug effects; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection / Q000379:methods
chemical_list: D007202:Indicators and Reagents; D008055:Lipids; C086724:Lipofectamine; D034741:RNA, Small Interfering; D004247:DNA
keywords: DNA; ES cells; Embryonic stem cells; Mouse; Plasmid; Transfection; siRNA