1S.C.-Y.W., Institute of Molecular Medicine, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan 333.
FASEB J. 2013 Nov;27(11):4429-43. doi: 10.1096/fj.12-223586. Epub 2013 Jul 29.
The piggyBac transposon is one of the most attractive nonviral tools for mammalian genome manipulations. Given that piggybac mobilizes in a "cut-and-paste" fashion, integrant remobilization could potentially damage the host genome. Here, we report a novel piggyBac transposon system with a series of recombinant transposases. We found that the transposition activity of wild-type (PBase) and hyperactive (hyPBase) piggyBac transposases can be significantly increased by peptide fusions in a cell-type dependent fashion, with the greatest change typically seen in mouse embryonic stem (ES) cells. The two most potent recombinant transposases, TPLGMH and ThyPLGMH, give a 9- and 7-fold increase, respectively, in the number of integrants in HEK293 compared with Myc-tagged PBase (MycPBase), and both display 4-fold increase in generating induced pluripotential stem cells. Interestingly, ThyPLGMH but not TPLGMH shows improved chromosomal excision activity (2.5-fold). This unique feature of TPLGMH provides the first evidence that integration activity of a transposase can be drastically improved without increasing its remobilization activity. Transposition catalyzed by ThyPLGMH is more random and occurs further from CpG islands than that catalyzed by MycPBase or TPLGMH. Our transposon system diversifies the mammalian genetic toolbox and provides a spectrum of piggyBac transposases that is better suited to different experimental purposes.
猪 bac 转座子是最有吸引力的非病毒工具之一，用于哺乳动物基因组操作。鉴于猪 bac 以“切 - 粘贴”的方式移动，整合子的重新移动可能会损害宿主基因组。在这里，我们报告了一种具有一系列重组转座酶的新型猪 bac 转座子系统。我们发现，野生型 (PBase) 和超活性 (hyPBase) 猪 bac 转座酶的转座活性可以通过肽融合以依赖于细胞类型的方式显著增加，在小鼠胚胎干细胞 (ES) 细胞中通常观察到最大的变化。两种最有效的重组转座酶 TPLGMH 和 ThyPLGMH，与 Myc 标记的 PBase (MycPBase) 相比，在 HEK293 中的整合子数量分别增加了 9 倍和 7 倍，并且都显示出诱导多能干细胞的 4 倍增加。有趣的是，只有 ThyPLGMH 而不是 TPLGMH 显示出改善的染色体切除活性（2.5 倍）。TPLGMH 的这一独特特征提供了第一个证据，即转座酶的整合活性可以在不增加其重新移动活性的情况下大大提高。由 ThyPLGMH 催化的转座更随机，并且发生在离 CpG 岛更远的地方，而不是由 MycPBase 或 TPLGMH 催化的转座。我们的转座子系统使哺乳动物遗传工具包多样化，并提供了一系列更适合不同实验目的的猪 bac 转座酶。
