title: Improved site-specific recombinase-based method to produce selectable marker- and vector-backbone-free transgenic cells.
abstract: PhiC31 integrase-mediated gene delivery has been extensively used in gene therapy and animal transgenesis. However, random integration events are observed in phiC31-mediated integration in different types of mammalian cells; as a result, the efficiencies of pseudo attP site integration and evaluation of site-specific integration are compromised. To improve this system, we used an attB-TK fusion gene as a negative selection marker, thereby eliminating random integration during phiC31-mediated transfection. We also excised the selection system and plasmid bacterial backbone by using two other site-specific recombinases, Cre and Dre. Thus, we generated clean transgenic bovine fetal fibroblast cells free of selectable marker and plasmid bacterial backbone. These clean cells were used as donor nuclei for somatic cell nuclear transfer (SCNT), indicating a similar developmental competence of SCNT embryos to that of non-transgenic cells. Therefore, the present gene delivery system facilitated the development of gene therapy and agricultural biotechnology.
authors.affiliation: 1] College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, PR China [2] Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling, Shaanxi, PR China [3].
authors.forename: Yuan
authors.initials: Y
authors.lastname: Yu
authors.affiliation: 1] College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, PR China [2] Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling, Shaanxi, PR China [3].
authors.forename: Qi
authors.initials: Q
authors.lastname: Tong
authors.affiliation: 1] College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, PR China [2] Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling, Shaanxi, PR China [3].
authors.forename: Zhongxia
authors.initials: Z
authors.lastname: Li
authors.affiliation: 1] College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, PR China [2] Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling, Shaanxi, PR China.
authors.forename: Jinhai
authors.initials: J
authors.lastname: Tian
authors.affiliation: 1] College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, PR China [2] Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling, Shaanxi, PR China.
authors.forename: Yizhi
authors.initials: Y
authors.lastname: Wang
authors.affiliation: 1] College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, PR China [2] Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling, Shaanxi, PR China.
authors.forename: Feng
authors.initials: F
authors.lastname: Su
authors.affiliation: 1] College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, PR China [2] Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling, Shaanxi, PR China.
authors.forename: Yongsheng
authors.initials: Y
authors.lastname: Wang
authors.affiliation: 1] College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, PR China [2] Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling, Shaanxi, PR China.
authors.forename: Jun
authors.initials: J
authors.lastname: Liu
authors.affiliation: 1] College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, PR China [2] Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling, Shaanxi, PR China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002478:Cells, Cultured; D005347:Fibroblasts / Q000502:physiology*; D018014:Gene Transfer Techniques; D005818:Genetic Engineering / Q000379:methods*; D005819:Genetic Markers / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D045522:Recombinases / Q000235:genetics*; D014162:Transfection / Q000379:methods*; D019076:Transgenes / Q000235:genetics*
chemical_list: D005819:Genetic Markers; D045522:Recombinases