title: Effectiveness of liposomes to transfect livestock fibroblasts.
abstract: The development of an efficient transfection system in livestock cells is an important step towards investigating gene transfer and the functioning and production of transgenic animals. Important factors involved in cationic liposome mediated gene transfer were evaluated through in vitro transfection of bovine, caprine and ovine fibroblast cells. Transfection of plasmid DNA complexes of different commercially available liposomes (Lipofectamine, Lipofectin, Cellfectin and DMRIE-C; Gibco-BRL, USA) was evaluated utilizing the following parameters: DNA/liposome ratio, cell density, DNA conformation, and the effect of transfection time on the efficiency of bovine fibroblasts to express a reporter gene. The effects and concentrations of liposomes were also evaluated in caprine and ovine fibroblasts. Lipofectamine alone and Lipofectamine with Plus reagent induced high-frequency expression of beta-galactosidase and neo genes in all cells evaluated (47 and 88.3%, respectively). Regarding phenotype, chromosomal stability was similar in transfected and non-transfected cells. The parameters set in this study will establish a foundation for utilizing transfected fibroblast cells to generate transgenic animals through nuclear transfer technology and gene function studies.
authors.affiliation: Laboratório de Transferência e Expressão de Genes, EMBRAPA Recursos Genéticos e Biotecnologia, Parque Estação Biológica, 70770-900 Brasília, DF, Brazil.
authors.forename: Raniere R
authors.initials: RR
authors.lastname: Oliveira
authors.forename: Daniela M de
authors.initials: DM
authors.lastname: Carvalho
authors.forename: Sharon
authors.initials: S
authors.lastname: Lisauskas
authors.forename: Eduardo
authors.initials: E
authors.lastname: Mello
authors.forename: Giovanni R
authors.initials: GR
authors.lastname: Vianna
authors.forename: Margot A N
authors.initials: MA
authors.lastname: Dode
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Rumpf
authors.forename: Francisco J L
authors.initials: FJ
authors.lastname: Aragão
authors.forename: Elíbio L
authors.initials: EL
authors.lastname: Rech
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D002417:Cattle / Q000235:genetics*; D002452:Cell Count; D002478:Cells, Cultured; D003587:Cytomegalovirus; D004247:DNA / Q000737:chemistry; D005347:Fibroblasts / Q000637:transplantation*; D015870:Gene Expression; D005822:Genetic Vectors; D008081:Liposomes; D010957:Plasmids / Q000235:genetics; D015203:Reproducibility of Results; D012756:Sheep / Q000235:genetics; D013552:Swine / Q000235:genetics; D014162:Transfection / Q000379:methods*; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D008081:Liposomes; D004247:DNA; D001616:beta-Galactosidase