Efficient gene transfer into murine embryonic stem cells by nucleofection

Authors:
Lorenz P, Harnack U and Morgenstern R
In:
Source: Biotechnol Lett
Publication Date: (2004)
Issue: 26(20): 1589-1592
Research Area:
Cancer Research/Cell Biology
Cells used in publication:
Embryonic stem cell (ES), mouse
Species: mouse
Tissue Origin: embryo
Platform:
Nucleofectorâ„¢ I/II/2b
Experiment
Transfection effciencies in viable mouse ES cells (murine embryonic stem cell line ES-D3 (ATCC)) were between 85% and 96% with high levels of EGFP expression [mean fluorescence intensity (MFI): 630 +/- 90] 24 h after nucleofection.
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.