Troyanovsky Boris, Bitko Vira, Pastukh Viktor, Fouty Brian, Solodushko Victor
Department of Pharmacology, University of South Alabama School of Medicine, Mobile, Alabama, USA.
Exscien Corporation, Mobile, Alabama, USA.
Mol Ther Nucleic Acids. 2016 Oct 4;5(10):e369. doi: 10.1038/mtna.2016.76.
Minimal piggyBac vectors are a modified single-plasmid version of the classical piggyBac delivery system that can be used for stable transgene integration. These vectors have a truncated terminal domain in the delivery cassette and thus, integrate significantly less flanking transposon DNA into host cell chromatin than classical piggyBac vectors. Herein, we test various characteristics of this modified transposon. The integration efficiency of minimal piggyBac vectors was inversely related to the size of both the transposon and the entire plasmid, but inserts as large as 15 kb were efficiently integrated. Open and super-coiled vectors demonstrated the same integration efficiency while DNA methylation decreased the integration efficiency and silenced the expression of previously integrated sequences in some cell types. Importantly, the incidence of plasmid backbone integration was not increased above that seen in nontransposon control vectors. In BALB/c mice, we demonstrated prolonged expression of two transgenes (intracellular mCherry and secretable Gaussia luciferase) when delivered by the minimal piggyBac that resulted in a more sustained antibody production against the immunogenic luciferase than when delivered by a transient (nontransposon) vector plasmid. We conclude that minimal piggyBac vectors are an effective alternative to other integrative systems for stable DNA delivery in vitro and in vivo.
微型猪尾巴病毒载体是经典猪尾巴病毒递送系统的一种改良单质粒版本，可用于稳定转基因整合。这些载体在递送盒中有一个截短的末端结构域，因此，与经典猪尾巴病毒载体相比，整合到宿主细胞染色质中的侧翼转座子DNA显著减少。在此，我们测试了这种改良转座子的各种特性。微型猪尾巴病毒载体的整合效率与转座子和整个质粒的大小呈负相关，但长达15 kb的插入片段能有效整合。开放型和超螺旋型载体表现出相同的整合效率，而DNA甲基化会降低整合效率，并使某些细胞类型中先前整合序列的表达沉默。重要的是，质粒骨架整合的发生率并未高于非转座子对照载体。在BALB/c小鼠中，我们证明了通过微型猪尾巴病毒载体递送时，两个转基因（细胞内的mCherry和可分泌的高斯荧光素酶）的表达时间延长，与通过瞬时（非转座子）载体质粒递送相比，产生了更持久的针对免疫原性荧光素酶的抗体。我们得出结论，微型猪尾巴病毒载体是体外和体内稳定DNA递送的其他整合系统的有效替代方案。
