title: Nucleofection of human embryonic stem cells.
abstract: Human embryonic stem (hES) cells provide an important tool for the study of human development, disease, and tissue regeneration. Technologies for efficient genetic modification are required to exploit hES cells fully for these applications. Here we present a customized protocol for the transfection of hES cells with the Nucleofector technology and compare its efficiency with conventional electroporation and lipofection. Cell survival and transfection efficiency were quantified using an enhanced green fluorescent protein (EGFP) reporter construct. Our optimized nucleofection parameters yielded survival rates >70%. Under these conditions, 66% of the surviving cells showed transgene expression 24 h after nucleofection. Transfected cells maintained expression of the pluripotency- associated markers Tra-1-60, Tra-1-81, and Oct4 and could be expanded to stably transgene-expressing clones. The low quantities of hES cells and DNA required for nucleofection could make this method an attractive tool for miniaturized high throughput screening (HTS) applications.
authors.affiliation: Institute of Reconstructive Neurobiology, University of Bonn Life & Brain Center and Hertie Foundation, 53105 Bonn, Germany.
authors.forename: Henrike
authors.initials: H
authors.lastname: Siemen
authors.forename: Michael
authors.initials: M
authors.lastname: Nix
authors.forename: Elmar
authors.initials: E
authors.lastname: Endl
authors.forename: Philipp
authors.initials: P
authors.lastname: Koch
authors.forename: Joseph
authors.initials: J
authors.lastname: Itskovitz-Eldor
authors.forename: Oliver
authors.initials: O
authors.lastname: Brüstle
mesh_terms: D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D002470:Cell Survival; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D018274:Electroporation; D004622:Embryo, Mammalian / Q000166:cytology*; D005347:Fibroblasts / Q000378:metabolism; D005434:Flow Cytometry; D015316:Genetic Therapy / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D013234:Stem Cells / Q000166:cytology*; D014162:Transfection / Q000379:methods*; D019076:Transgenes
chemical_list: D049452:Green Fluorescent Proteins; D004247:DNA