title: Efficient transfection of primarily cultured porcine embryonic fibroblasts using the Amaxa Nucleofection system.
abstract: Porcine embryonic fibroblasts (PEF) are important as donor cells for nuclear transfer for generation of genetically modified pigs. In this study, we determined an optimal protocol for transfection of PEF with the Amaxa Nucleofection system, which directly transfers DNA into the nucleus of cells, and compared its efficiency with conventional lipofection and electroporation. Cell survival and transfection efficiency were assessed using dye-exclusion assay and a green fluorescent protein (GFP) reporter construct, respectively. Our optimized nucleofection parameters yielded survival rates above 60%. Under these conditions, FACS analysis demonstrated that 79% of surviving cells exhibited transgene expression 48 h after nucleofection when program U23 was used. This efficiency was higher than that of transfection of PEFs with electroporation (ca. 3-53%) or lipofection (ca. 3-8%). Transfected cells could be expanded as stably transgene-expressing clones over a month. When porcine nuclear transfer (NT) was performed using stable transformant expressing GFP as a donor cell, 5-6% of reconstituted embryos developed to blastocysts, from which 30-50% of embryos exhibited NT-embryo-derived green fluorescence. Under the conditions evaluated, nucleofection exhibited higher efficiency than conventional electroporation and lipofection, and may be a useful alternative for generation of genetically engineered pigs through nuclear transfer.
authors.affiliation: Laboratory of Frontier Medicine, Frontier Science Research Center, Kagoshima University, Kagoshima, Japan.
authors.forename: Asuka
authors.initials: A
authors.lastname: Nakayama
authors.forename: Masahiro
authors.initials: M
authors.lastname: Sato
authors.forename: Mariko
authors.initials: M
authors.lastname: Shinohara
authors.forename: Shyuichiro
authors.initials: S
authors.lastname: Matsubara
authors.forename: Takaaki
authors.initials: T
authors.lastname: Yokomine
authors.forename: Eri
authors.initials: E
authors.lastname: Akasaka
authors.forename: Mitsutoshi
authors.initials: M
authors.lastname: Yoshida
authors.forename: Sonshin
authors.initials: S
authors.lastname: Takao
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000166:cytology; D018929:Cell Culture Techniques / Q000379:methods*; D002469:Cell Separation; D019976:Cloning, Organism / Q000379:methods*; D004247:DNA / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D018014:Gene Transfer Techniques; D014644:Genetic Variation; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D053652:Nuclear Transfer Techniques; D013552:Swine; D014162:Transfection / Q000379:methods*
chemical_list: D049452:Green Fluorescent Proteins; D004247:DNA