An optimized, broadly applicable piggyBac transposon induction system.
An optimized, broadly applicable piggyBac transposon induction system.
复制标题
DOI：
10.1093/nar/gkw1290
复制
发表时间：
2017-04-20
影响因子：
14.9
通讯作者：
Mitra RD
中科院分区：
文献类型：
--
作者：
Qi Z;Wilkinson MN;Chen X;Sankararaman S;Mayhew D;Mitra RD
piggyBac（PB）转座子已用于许多生物学应用中。将PB转座子插入基因组可以破坏基因或调控区，影响细胞功能，因此对于许多实验来说，PB转座受到严格控制是很重要的。在这里，我们系统地描述了三种方法的PB转座子在四个细胞系的翻译后控制。我们研究了PB转座酶与ERT 2和两个降解结构域（FKBP-DD、DHFR-DD）在多个方向上的融合，并确定了（i）实现的诱导倍数，（ii）活化构建体的绝对转座效率和（iii）两种诱导分子对细胞转录和功能的影响。我们发现，FKBP-DD赋予PB转座酶更高的转座活性和更好的动态范围比可以实现与其他系统。此外，我们发现FKBP-DD以可逆和剂量依赖性的方式调节转座子活性。最后，我们表明FKBP-DD的化学诱导剂Shld 1不会干扰干细胞分化，而他莫昔芬具有显着的作用。我们相信，基于FKBP的PB转座子诱导将是有用的转座子介导的基因组工程，插入诱变和转录因子结合的全基因组定位。
The piggyBac (PB) transposon has been used in a number of biological applications. The insertion of PB transposons into the genome can disrupt genes or regulatory regions, impacting cellular function, so for many experiments it is important that PB transposition is tightly controlled. Here, we systematically characterize three methods for the post-translational control of the PB transposon in four cell lines. We investigated fusions of the PB transposase with ERT2 and two degradation domains (FKBP-DD, DHFR-DD), in multiple orientations, and determined (i) the fold-induction achieved, (ii) the absolute transposition efficiency of the activated construct and (iii) the effects of two inducer molecules on cellular transcription and function. We found that the FKBP-DD confers the PB transposase with a higher transposition activity and better dynamic range than can be achieved with the other systems. In addition, we found that the FKBP-DD regulates transposon activity in a reversible and dose-dependent manner. Finally, we showed that Shld1, the chemical inducer of FKBP-DD, does not interfere with stem cell differentiation, whereas tamoxifen has significant effects. We believe the FKBP-based PB transposon induction will be useful for transposon-mediated genome engineering, insertional mutagenesis and the genome-wide mapping of transcription factor binding.