The Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA.
Transgenic Res. 2011 Oct;20(5):1125-37. doi: 10.1007/s11248-010-9481-7. Epub 2011 Jan 9.
Swine transgenesis by pronuclear injection or cloning has traditionally relied on illegitimate recombination of DNA into the pig genome. This often results in animals containing concatemeric arrays of transgenes that complicate characterization and can impair long-term transgene stability and expression. This is inconsistent with regulatory guidance for transgenic livestock, which also discourages the use of selection markers, particularly antibiotic resistance genes. We demonstrate that the Sleeping Beauty (SB) transposon system effectively delivers monomeric, multi-copy transgenes to the pig embryo genome by pronuclear injection without markers, as well as to donor cells for founder generation by cloning. Here we show that our method of transposon-mediated transgenesis yielded 38 cloned founder pigs that altogether harbored 100 integrants for five distinct transposons encoding either human APOBEC3G or YFP-Cre. Two strategies were employed to facilitate elimination of antibiotic genes from transgenic pigs, one based on Cre-recombinase and the other by segregation of independently transposed transgenes upon breeding.
猪的原核注射或克隆转基因技术传统上依赖于 DNA 与猪基因组的非合法重组。这通常导致动物含有串联的转基因,这使得其特征复杂化,并可能损害长期转基因的稳定性和表达。这与转基因牲畜的监管指南不一致,该指南也不鼓励使用选择标记,特别是抗生素抗性基因。我们证明,Sleeping Beauty(SB)转座子系统通过原核注射有效地将单体、多拷贝的转基因传递到猪胚胎基因组中,而无需标记,也可用于克隆生成供体细胞。在这里,我们展示了我们的转座子介导的转基因方法产生了 38 头克隆的起始猪,它们总共携带了 5 种不同的转座子的 100 个整合子,这些转座子编码人 APOBEC3G 或 YFP-Cre。我们采用了两种策略来促进从转基因猪中消除抗生素基因,一种基于 Cre 重组酶,另一种则通过在繁殖时独立转座的转基因的分离来实现。