title: In vitro development of bovine nuclear transfer embryos from transgenic clonal lines of adult and fetal fibroblast cells of the same genotype.
abstract: This study examined bovine cloning strategies that may be used for gene targeting in animals of known phenotypic traits. Fibroblast cells derived from an adult and a fetus of the same genotype were transfected with a plasmid (pEGFP-N1) containing the enhanced green fluorescence protein and neomycin-resistant genes. After transfecting 2 x 10(5) cells, 49 adult and 35 fetal cell colonies were obtained. Green fluorescence expression was observed in 35 out of 49 (71.4%) adult clones and in 30 out of 35 (85.7%) fetal clones. Developmental rates to the blastocyst stage following nuclear transfer (NT) did not differ among nontransfected cell lines (adult, 20.0%; NT fetal, 18.3%), whereas developmental rates were significantly lower for adult and fetal cell lines expressing enhanced green fluorescent protein (EGFP; 11.3% and 6.4%, respectively, P < 0.05). However, there was no decrease in NT developmental rates (19.8%) when donor nuclei from EGFP-transfected cell lines not expressing EGFP but retaining neomycin-resistant gene expression were used as donor nuclei. NT embryos from adult and fetal cell lines had similar morphology, cell number, and ploidy. The results indicated that adult and NT fetal cells (identical genotype) can complete clonal propagation, including transfection and selection, and can be used to produce transgenic NT embryos; however, a possible deleterious effect of EGFP on embryo development should be considered in future gene targeting studies.
authors.affiliation: Department of Animal and Dairy Science, University of Georgia, Athens, Georgia 30602, USA.
authors.forename: Sezen
authors.initials: S
authors.lastname: Arat
authors.forename: John
authors.initials: J
authors.lastname: Gibbons
authors.forename: S Jacek
authors.initials: SJ
authors.lastname: Rzucidlo
authors.forename: Donald S
authors.initials: DS
authors.lastname: Respess
authors.forename: Monica
authors.initials: M
authors.lastname: Tumlin
authors.forename: Steven L
authors.initials: SL
authors.lastname: Stice
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D002417:Cattle / Q000196:embryology*; D019976:Cloning, Organism; D005314:Embryonic and Fetal Development; D005347:Fibroblasts / Q000378:metabolism / Q000648:ultrastructure*; D015870:Gene Expression; D005838:Genotype; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000648:ultrastructure; D011003:Ploidies; D011994:Recombinant Proteins; D014162:Transfection
chemical_list: D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins