title: A novel method for the production of transgenic cloned pigs: electroporation-mediated gene transfer to non-cultured cells and subsequent selection with puromycin.
abstract: Puromycin N-acetyl transferase gene (pac), of which the gene product catalyzes antibiotic puromycin (an effective inhibitor of protein synthesis), has been widely used as a dominant selection marker in embryonic stem (ES) cell-mediated transgenesis. The present study is the first to report on the usefulness of puromycin for production of enhanced green fluorescent protein (EGFP) transgenic piglets after somatic cell cloning and embryo transfer. Somatic cells isolated from porcine fetuses at 73 days of gestation were immediately electroporated with a transgene (pCAG-EGFPac) carrying both EGFP cDNA and pac. This procedure aims to avoid aging effects thought to be generated during cell culture. The recombinant cells were selected with puromycin at a low concentration (2 microg/ml), cultured for 7 days, and then screened for EGFP expression before somatic cell cloning. The manipulated embryos were transplanted into the oviducts of 14 foster mother sows. Four of the foster sows became pregnant and nine piglets were delivered. Of the nine piglets, eight died shortly after birth and one grew healthy after weaning. Results indicate that puromycin can be used for the selection of recombinant cells from noncultured cells, and moreover, may confer the production of genetically engineered newborns via nuclear transfer techniques in pigs.
authors.affiliation: Department of Developmental Biology, Division of Insect and Animal, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, 305-0901, Japan. kettle@affrc.go.jp
authors.forename: Satoshi
authors.initials: S
authors.lastname: Watanabe
authors.forename: Masaki
authors.initials: M
authors.lastname: Iwamoto
authors.forename: Shun-ichi
authors.initials: S
authors.lastname: Suzuki
authors.forename: Daiichiro
authors.initials: D
authors.lastname: Fuchimoto
authors.forename: Daisuke
authors.initials: D
authors.lastname: Honma
authors.forename: Takashi
authors.initials: T
authors.lastname: Nagai
authors.forename: Michiko
authors.initials: M
authors.lastname: Hashimoto
authors.forename: Satoko
authors.initials: S
authors.lastname: Yazaki
authors.forename: Masahiro
authors.initials: M
authors.lastname: Sato
authors.forename: Akira
authors.initials: A
authors.lastname: Onishi
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D000963:Antimetabolites / Q000494:pharmacology*; D015153:Blotting, Western; D002467:Cell Nucleus / Q000235:genetics; D002477:Cells; D019976:Cloning, Organism; D004247:DNA / Q000235:genetics; D019008:Drug Resistance, Neoplasm; D018274:Electroporation; D005260:Female; D005333:Fetus / Q000166:cytology; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D011247:Pregnancy; D011691:Puromycin / Q000494:pharmacology*; D013552:Swine; D014162:Transfection; D019076:Transgenes / Q000235:genetics
chemical_list: D000963:Antimetabolites; D049452:Green Fluorescent Proteins; D011691:Puromycin; D004247:DNA