Institut für Nutztiergenetik, Friedrich-Loeffler-Institut, Neustadt, Germany.
PLoS One. 2011;6(8):e23573. doi: 10.1371/journal.pone.0023573. Epub 2011 Aug 29.
Genetic engineering can expand the utility of pigs for modeling human diseases, and for developing advanced therapeutic approaches. However, the inefficient production of transgenic pigs represents a technological bottleneck. Here, we assessed the hyperactive Sleeping Beauty (SB100X) transposon system for enzyme-catalyzed transgene integration into the embryonic porcine genome. The components of the transposon vector system were microinjected as circular plasmids into the cytoplasm of porcine zygotes, resulting in high frequencies of transgenic fetuses and piglets. The transgenic animals showed normal development and persistent reporter gene expression for >12 months. Molecular hallmarks of transposition were confirmed by analysis of 25 genomic insertion sites. We demonstrate germ-line transmission, segregation of individual transposons, and continued, copy number-dependent transgene expression in F1-offspring. In addition, we demonstrate target-selected gene insertion into transposon-tagged genomic loci by Cre-loxP-based cassette exchange in somatic cells followed by nuclear transfer. Transposase-catalyzed transgenesis in a large mammalian species expands the arsenal of transgenic technologies for use in domestic animals and will facilitate the development of large animal models for human diseases.
基因工程可以扩大猪在模拟人类疾病和开发先进治疗方法方面的应用。然而，转基因猪的低效生产是一个技术瓶颈。在这里，我们评估了超活性 Sleeping Beauty (SB100X) 转座子系统，用于酶促转基因整合到胚胎猪基因组中。转座子载体系统的组件作为环状质粒微注射到猪受精卵的细胞质中，导致转基因胎儿和仔猪的高频率出现。转基因动物表现出正常的发育和持续超过 12 个月的报告基因表达。通过分析 25 个基因组插入位点证实了转座的分子特征。我们证明了生殖系传递、单个转座子的分离以及 F1 后代中持续的、依赖拷贝数的转基因表达。此外，我们通过体细胞中的 Cre-loxP 基于盒式交换证明了目标选择基因插入到转座子标记的基因组位点，然后进行核转移。在大型哺乳动物物种中转座酶催化的转基因技术扩展了用于家畜的转基因技术的武器库，并将促进人类疾病的大型动物模型的开发。
