title: Mouse embryonic stem cells efficiently lipofected with nuclear localization peptide result in a high yield of chimeric mice and retain germline transmission potency.
abstract: Embryonic stem (ES) cells are an important tool in developmental biology, genomics, and transgenic methods, as well as in potential clinical applications such as gene therapy or tissue engineering. Electroporation is the standard transfection method for mouse ES (mES) cells because lipofection is quite inefficient. It is also unclear if mES cells treated with cationic lipids maintain pluripotency. We have developed a simple lipofection method for high efficiency transfection and stable transgene expression by employing the nonclassical nuclear localization signal M9 derived from the heterogeneous nuclear ribonucleoprotein A1. In contrast to using 20 microg DNA for 10 x 10(6) cells via electroporation which resulted in 10-20 positive cells/mm2, M9-assisted lipofection of 2 x 10(5) cells with 2 microg DNA resulted in > 150 positive cells/mm2. Electroporation produced only 0.16% EGFP positive cells with fluorescence intensity (FI) > 1000 by FACS assay, while M9-lipofection produced 36-fold more highly EGFP positive cells (5.75%) with FI > 1000. Using 2.5 x 10(6) ES cells and 6 microg linearized DNA followed by selection with G418, electroporation yielded 17 EGFP expressing colonies, while M9-assisted lipofection yielded 72 EGFP expressing colonies. The mES cells that stably expressed EGFP following M9-assisted lipofection yielded > 66% chimeric mice (8 of 12) and contributed efficiently to the germline. In an example of gene targeting, a knock-in mouse was produced from an ES clone screened from 200 G418-resistant colonies generated via M9-assisted lipofection. To our knowledge, this is the first report of generation of transgenic or knock-in mice obtained from lipofected mES cells and this method may facilitate large scale genomic studies of ES developmental biology or large scale generation of mouse models of human disease.
authors.affiliation: Department of Chemical Engineering, Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: Haiching
authors.initials: H
authors.lastname: Ma
authors.forename: Qin
authors.initials: Q
authors.lastname: Liu
authors.forename: Scott L
authors.initials: SL
authors.lastname: Diamond
authors.forename: Eric A
authors.initials: EA
authors.lastname: Pierce
mesh_terms: D000818:Animals; D002678:Chimera; D018274:Electroporation; D004622:Embryo, Mammalian / Q000166:cytology*; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000737:chemistry*; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D008055:Lipids / Q000737:chemistry; D008081:Liposomes; D051379:Mice / Q000196:embryology / Q000235:genetics*; D008822:Mice, Transgenic; D019913:Nuclear Localization Signals; D010446:Peptide Fragments / Q000737:chemistry*; D021382:Protein Sorting Signals; D012261:Ribonucleoproteins / Q000235:genetics; D013234:Stem Cells / Q000378:metabolism* / Q000648:ultrastructure; D014162:Transfection / Q000379:methods*
chemical_list: D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D036341:Intercellular Signaling Peptides and Proteins; D008055:Lipids; C086724:Lipofectamine; D008081:Liposomes; C494045:M9 nuclear localization signal peptide; D019913:Nuclear Localization Signals; D010446:Peptide Fragments; D021382:Protein Sorting Signals; D012261:Ribonucleoproteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins