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Fast and Efficient Transfection of Mouse Embryonic Stem Cells Using Non-Viral Reagents
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. GE Healthcare BioSci AB, Bjorkgatan 30, SE-75184 Uppsala, Sweden..
2016 (English)In: Stem Cell Reviews, ISSN 1550-8943, E-ISSN 1558-6804, Vol. 12, no 5, 584-591 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2016. Vol. 12, no 5, 584-591 p.
Keyword [en]
Transfection, Mouse, Embryonic stem cells, ES cells, siRNA, DNA, Plasmid
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-307280DOI: 10.1007/s12015-016-9673-5ISI: 000385138500009PubMedID: 27358240OAI: oai:DiVA.org:uu-307280DiVA: diva2:1046316
Available from: 2016-11-14 Created: 2016-11-11 Last updated: 2016-11-14Bibliographically approved

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Kadekar, SandeepPijuan-Galito, SaraAnnerén, Cecilia
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