title: Efficient gene transfer into murine embryonic stem cells by 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.
authors.affiliation: Institute of Pharmacology and Toxicology, Charité Universitätsmedizin Berlin, Berlin, Germany.
authors.forename: Peer
authors.initials: P
authors.lastname: Lorenz
authors.forename: Ulf
authors.initials: U
authors.lastname: Harnack
authors.forename: Rudolf
authors.initials: R
authors.lastname: Morgenstern
mesh_terms: D000818:Animals; D002467:Cell Nucleus / Q000235:genetics*; D002470:Cell Survival; D002478:Cells, Cultured; D018014:Gene Transfer Techniques; D005818:Genetic Engineering / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics*; D051379:Mice; D013234:Stem Cells / Q000502:physiology*; D014162:Transfection / Q000379:methods*
chemical_list: D049452:Green Fluorescent Proteins