title: Transgene expression of green fluorescent protein and germ line transmission in cloned pigs derived from in vitro transfected adult fibroblasts.
abstract: The pig represents the xenogeneic donor of choice for future organ transplantation in humans for anatomical and physiological reasons. However, to bypass several immunological barriers, strong and stable human genes expression must occur in the pig's organs. In this study we created transgenic pigs using in vitro transfection of cultured cells combined with somatic cell nuclear transfer (SCNT) to evaluate the ubiquitous transgene expression driven by pCAGGS vector in presence of different selectors. pCAGGS confirmed to be a very effective vector for ubiquitous transgene expression, irrespective of the selector that was used. Green fluorescent protein (GFP) expression observed in transfected fibroblasts was also maintained after nuclear transfer, through pre- and postimplantation development, at birth and during adulthood. Germ line transmission without silencing of the transgene was demonstrated. The ubiquitous expression of GFP was clearly confirmed in several tissues including endothelial cells, thus making it a suitable vector for the expression of multiple genes relevant to xenotransplantation where tissue specificity is not required. Finally cotransfection of green and red fluorescence protein transgenes was performed in fibroblasts and after nuclear transfer blastocysts expressing both fluorescent proteins were obtained.
authors.affiliation: Laboratorio di Tecnologie della Riproduzione, CIZ srl, Istituto Sperimentale Italiano Lazzaro Spallanzani, Cremona, Italy.
authors.forename: Dario
authors.initials: D
authors.lastname: Brunetti
authors.forename: Andrea
authors.initials: A
authors.lastname: Perota
authors.forename: Irina
authors.initials: I
authors.lastname: Lagutina
authors.forename: Silvia
authors.initials: S
authors.lastname: Colleoni
authors.forename: Roberto
authors.initials: R
authors.lastname: Duchi
authors.forename: Fiorella
authors.initials: F
authors.lastname: Calabrese
authors.forename: Michela
authors.initials: M
authors.lastname: Seveso
authors.forename: Emanuele
authors.initials: E
authors.lastname: Cozzi
authors.forename: Giovanna
authors.initials: G
authors.lastname: Lazzari
authors.forename: Franco
authors.initials: F
authors.lastname: Lucchini
authors.forename: Cesare
authors.initials: C
authors.lastname: Galli
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics / Q000378:metabolism*; D001755:Blastocyst / Q000378:metabolism; D002478:Cells, Cultured; D019976:Cloning, Organism; D004624:Embryo Transfer; D004622:Embryo, Mammalian / Q000378:metabolism*; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D005854:Germ Cells / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000096:biosynthesis*; D008297:Male; D013552:Swine / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D019076:Transgenes / Q000235:genetics
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins