title: Production of porcine cloned embryos derived from cells conditionally expressing an exogenous gene using Cre-loxP.
abstract: It is increasingly evident that conditional gene expression in pigs is necessary to make transgenic models. In this study, we investigated conditional expression in porcine fetal fibroblasts using Cre-loxP recombination, a system that has had limited application in large animals to date. Transformed fibroblasts were reprogrammed in enucleated oocytes to support further early embryonic development. Fetal fibroblasts from miniature pigs were used for transfection with a plasmid that contained a red fluorescent protein marker (pCALNL-DsRed) and a floxed neomycin-resistance gene. Cells were selected with 750 μg/ml neomycin for 2 weeks following transfection but did not express DsRed after visualization under a fluorescence microscope. Expression was achieved only after transient transfection with plasmid DNA that expressed the Cre recombinase enzyme. The cells that expressed DsRed were used for somatic cell nuclear transfer (SCNT). A total of 121 oocytes were used for SCNT and 76 cloned embryos (62.8%) were seen to have cleaved. Six blastocysts developed after SCNT and expressed DsRed. Deletion of the floxed neomycin-resistance gene was confirmed by reverse transcription polymerase chain reaction (RT-PCR) in cloned blastocysts. This study demonstrated that Cre-loxP recombination can be conducted successfully in miniature pig fibroblasts and that the sequentially transformed cells can develop to the pre-implantation embryo stage via SCNT.
authors.affiliation: Department of Theriogenology and Biotechnology, College of Veterinary Medicine and the Research Institute for Veterinary Science, Seoul National University, Seoul, Republic of Korea.
authors.forename: JoonHo
authors.initials: J
authors.lastname: Moon
authors.forename: SuJin
authors.initials: S
authors.lastname: Kim
authors.forename: HeeJung
authors.initials: H
authors.lastname: Park
authors.forename: JungTaek
authors.initials: J
authors.lastname: Kang
authors.forename: SolJi
authors.initials: S
authors.lastname: Park
authors.forename: OkJae
authors.initials: O
authors.lastname: Koo
authors.forename: Begona Roibas
authors.initials: BR
authors.lastname: da Torre
authors.forename: Islam M
authors.initials: IM
authors.lastname: Saadeldin
authors.forename: ByeongChun
authors.initials: B
authors.lastname: Lee
authors.forename: Goo
authors.initials: G
authors.lastname: Jang
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D019976:Cloning, Organism; D004622:Embryo, Mammalian / Q000378:metabolism*; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D015870:Gene Expression; D019426:Integrases / Q000235:genetics* / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D013552:Swine / Q000196:embryology*; D000097573:Red Fluorescent Protein
chemical_list: D019426:Integrases; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein; C045073:Cre recombinase