Alessio Ana P, Fili Alejandro E, Garrels Wiebke, Forcato Diego O, Olmos Nicotra María F, Liaudat Ana C, Bevacqua Romina J, Savy Virginia, Hiriart María I, Talluri Thirumala R, Owens Jesse B, Ivics Zoltán, Salamone Daniel F, Moisyadi Stefan, Kues Wilfried A, Bosch Pablo
Departamento de Biología Molecular, Facultad de Ciencias Exactas, Fco-Qcas y Naturales, Universidad Nacional de Río Cuarto, Córdoba, República Argentina.
Department of Biotechnology, Friedrich-Loeffler-Institut, Institut für Nutztiergenetik, Neustadt, Germany.
Theriogenology. 2016 Apr 15;85(7):1297-311.e2. doi: 10.1016/j.theriogenology.2015.12.016. Epub 2015 Dec 29.
Transposon-mediated transgenesis is a well-established tool for genome modification in small animal models. However, translation of this active transgenic method to large animals warrants further investigations. Here, the piggyBac (PB) and sleeping beauty (SB) transposon systems were assessed for stable gene transfer into the cattle genome. Bovine fibroblasts were transfected either with a helper-independent PB system or a binary SB system. Both transposons were highly active in bovine cells increasing the efficiency of DNA integration up to 88 times over basal nonfacilitated integrations in a colony formation assay. SB transposase catalyzed multiplex transgene integrations in fibroblast cells transfected with the helper vector and two donor vectors carrying different transgenes (fluorophore and neomycin resistance). Stably transfected fibroblasts were used for SCNT and on in vitro embryo culture, morphologically normal blastocysts that expressed the fluorophore were obtained with both transposon systems. The data indicate that transposition is a feasible approach for genetic engineering in the cattle genome.
转座子介导的转基因技术是一种在小动物模型中用于基因组修饰的成熟工具。然而，将这种活跃的转基因方法应用于大型动物仍需进一步研究。在此，对猪尾巴（PB）和睡美人（SB）转座子系统进行了评估，以确定其将基因稳定导入牛基因组的能力。使用非依赖辅助质粒的PB系统或二元SB系统转染牛成纤维细胞。在集落形成试验中，两种转座子在牛细胞中均具有高活性，使DNA整合效率比基础非辅助整合提高了88倍。SB转座酶在转染了辅助载体和携带不同转基因（荧光团和新霉素抗性）的两个供体质粒的成纤维细胞中催化了多转基因整合。将稳定转染的成纤维细胞用于体细胞核移植和体外胚胎培养，两种转座子系统均获得了表达荧光团的形态正常的囊胚。数据表明，转座是牛基因组基因工程的一种可行方法。
