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].
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.
Sci Rep. 2014 Feb 28;4:4240. doi: 10.1038/srep04240.
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.
PhiC31 整合酶介导的基因传递已被广泛应用于基因治疗和动物转基因。然而，在不同类型的哺乳动物细胞中，PhiC31 介导的整合会观察到随机整合事件；因此，假 attP 位点整合的效率和位点特异性整合的评估受到影响。为了改进该系统，我们使用 attB-TK 融合基因作为负选择标记，从而在 PhiC31 介导的转染过程中消除随机整合。我们还使用另外两种位点特异性重组酶 Cre 和 Dre 切除了选择系统和质粒细菌骨架。因此，我们生成了不含选择标记和质粒细菌骨架的清洁转基因牛胎儿成纤维细胞。这些清洁细胞被用作体细胞核移植 (SCNT) 的供体核，表明 SCNT 胚胎的发育能力与非转基因细胞相似。因此，该基因传递系统促进了基因治疗和农业生物技术的发展。
