Abstract
Genetic manipulation of embryonic stem (ES) cells is performed by non-viral as well as viral transfection methods. We tested the recently developed nucleofection method delivering plasmid DNA directly into the nucleus for the introduction of a plasmid encoding enhanced green fluorescent protein (EGFP) into murine ES cells. Cell viability decreased from 77% before to 40% 24 h after nucleofection. Transfection effciencies in viable stem cells were between 85% and 96% with high levels of EGFP expression [mean fluorescence intensity (MFI): 630 ± 90] 24 h after nucleofection. After a two week culture in geneticin (G418) selection medium, nearly 50% of the stem cells were EGFP positive and continued transgene expression (MFIs: 120–240) for a two further weeks. We conclude that nucleofection is an efficient nonviral gene transfer method for the introduction of genes into murine ES cells.
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References
Asahara T, Kalka C, Isner JM (2000) Stem cell therapy and gene transfer for regeneration. Gene Ther. 7: 451–457.
Blau HM, Brazelton TR, Weimann JM(2001) The evolving concept of a stem cell entity or function? Cell 105: 829–841.
Chung S, Sonntag KC, Andersson T, Bjorklund LM, Park JJ, Kim DW, Kang UJ, Isacson O, Kim KS (2002a) Genetic engineering of mouse embryonic stem cells by Nurr1 enhances differentiation and maturation into dopaminergic neurons. Eur. J. Neurosci. 16: 1829–1838.
Chung S, Andersson T, Sonntag KC, Bjorklund L, Isacson O, Kim KS et al. (2002b) Analysis of different promoter systems for efficient transgene expression in mouse embryonic stem cell lines. Stem Cells 20: 139–145.
Kim KS, Kim CH, Hwang DY, Seo H, Chung S, Hong SJ, Lim JK, Andersson T, Isacson O (2003) Orphan nuclear receptor Nurr1 directly transactivates the promoter activity of the tyrosine hydroxylase gene in a cell-specific manner. J Neurochem. 85: 622–634.
Ma Y, Ramezani A, Lewis R, Hawley RG, Thomson JA (2003) High-level sustained transgene expression in human embryonic stem cells using lentiviral vectors. Stem Cells 21: 111–117.
Martinet W, Schrijvers DM, Kochx MM (2003) Nucleofection as an efficient nonviral transfection method for human monocytic cells. Biotechnol. Lett. 25: 1025–1029.
Smith-Arica JR, Thomson AJ, Ansell R, Chiorini J, Davidson B, McWhir J (2003) Infection efficiency of human and mouse embryonic stem cells using adenoviral and adeno-associated viral vectors. Cloning Stem Cells 5: 51–62.
Templeton NS, Roberts DD, Safer B (1997) Efficient gene targeting in mouse embryonic stem cells. Gene Ther. 4: 700–709.
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Lorenz, P., Harnack, U. & Morgenstern, R. Efficient gene transfer into murine embryonic stem cells by nucleofection. Biotechnology Letters 26, 1589–1592 (2004). https://doi.org/10.1023/B:BILE.0000045658.33723.d6
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DOI: https://doi.org/10.1023/B:BILE.0000045658.33723.d6