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Efficient gene transfer into murine embryonic stem cells by nucleofection.

Peer Lorenz·Ulf Harnack·Rudolf Morgenstern
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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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AnimalsCell NucleusCell SurvivalCells, CulturedGene Transfer TechniquesGenetic EngineeringGreen Fluorescent ProteinsMiceStem CellsTransfectionJournal Article

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Peer Lorenz, Ulf Harnack, Rudolf Morgenstern. 2004. Efficient gene transfer into murine embryonic stem cells by nucleofection.. https://doi.org/10.1023/b%3Abile.0000045658.33723.d6

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