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Messenger RNA electroporation is highly efficient in mouse embryonic stem cells: successful FLPe- and Cre-mediated recombination

Gene Therapy volume 11, pages 1606–1610 (2004)Cite this article

Abstract

Development of efficient short-term gene transfer technologies for embryonic stem (ES) cells is urgently needed for various existing and new ES cell-based research strategies. In this study, we present a highly efficient, nonviral non-DNA technology for genetic loading of mouse ES cells based on electroporation of defined mRNA. Here, we show that mouse ES cells can be efficiently loaded with mRNA encoding a green fluorescent reporter protein, resulting in a level of at least 90% of transgene expression without loss of cell viability and phenotype. To show that transgenes, introduced by mRNA electroporation, exert a specific cellular function in transfected cells, we electroporated stably transfected ES cell lines with mRNA encoding FLPe or Cre recombinase proteins in order to excise an FRT- or LoxP-flanked reporter gene. The results, as determined by the disappearance and/or appearance of a fluorescent reporter gene expression, show that FLPe and Cre recombinase proteins, introduced by mRNA electroporation, efficiently exert their function without influence on further culture of undifferentiated ES cell populations and their ability to differentiate towards a specific lineage.

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Acknowledgements

Part of this work was undertaken during a GERON-funded visit by Peter Ponsaerts to the Singh Laboratory. We are indebted to Geron Corporation (Palo Alto, CA, USA) for their support. We thank Dr Karen May from the Singh Laboratory for additional flow cytometric analysis. We also acknowledge support from Grant No. WO.012.02.N (Scientific Research Community FWO) and Grant No. 7.0004.03 (FWO-Levenslijn) of the fund for Scientific Research-Flanders (FWO-Vlaanderen). LVDE holds a PhD fellowship from the Institute for Science and Technology (IWT). VFIVT is a postdoctoral fellow of the FWO-Vlaanderen. We thank Professor Christine van Broeckhoven and Bart De Vil (Transgene Core University of Antwerp) for assistance with ES cell culture. This work was also supported within the frame of the Inter-University Attraction poles (IUAP) program P5/19 of the Belgian Federal Science Policy Office.

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  1. P Ponsaerts, JP Brown and D Van den Plas: These authors contributed equally and should be regarded as joint first authors

Authors and Affiliations

  1. Laboratory of Experimental Hematology, Faculty of Medicine, University of Antwerp, Antwerp University Hospital, Antwerp, Belgium

    P Ponsaerts, L Van den Eeden, DR Van Bockstaele, VFI Van Tendeloo & ZN Berneman

  2. Laboratory of Nuclear Reprogramming, Roslin Institute (Edinburgh), Midlothian, Scotland, UK

    JP Brown & PB Singh

  3. Department of Biomedical Sciences, Laboratory of Molecular Biotechnology, University of Antwerp, Antwerp, Belgium

    D Van den Plas & J Merregaert

  4. Department of Clinical Pharmacotherapy, Faculty of Medicine, University of Antwerp, Antwerp University Hospital, Antwerp, Belgium

    PG Jorens

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  1. P Ponsaerts
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  2. JP Brown
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  10. ZN Berneman
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Ponsaerts, P., Brown, J., Van den Plas, D. et al. Messenger RNA electroporation is highly efficient in mouse embryonic stem cells: successful FLPe- and Cre-mediated recombination. Gene Ther 11, 1606–1610 (2004). https://doi.org/10.1038/sj.gt.3302342

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