Mátés Lajos
Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary.
Methods Mol Biol. 2011;738:87-99. doi: 10.1007/978-1-61779-099-7_6.
DNA-based transposons are natural gene delivery vehicles. Similarly to retroviruses, these elements -integrate into the chromosomes of host cells, but their life-cycle does not involve reverse transcription and they are not infectious. Transposon-based gene delivery has several advantageous features compared to viral methods; however, its efficacy has been the bottleneck of transposon utilization. Recently, using a novel strategy for in vitro evolution, we created a new hyperactive version (SB100X) of the vertebrate-specific Sleeping Beauty (SB) transposase. SB100X, when coupled with enhanced inverted terminal repeat structure T2 type SB transposons, is over 100-fold more active in mammalian cells than the prototype. We established protocol for SB100X mediated rodent transgenesis resulting on the average 35% transgenic founders with a low average number (1-2) of transgene insertions per founder. Due to these characteristics the SB100X based protocol opens the possibility of designing SB based transgenes also for in vivo knockdown experiments. By the same token, single copy transgene units introduced by the SB transposon system, more than being less prone to transgene silencing, also allow better control of transgene expression levels and patterns.
基于DNA的转座子是天然的基因传递载体。与逆转录病毒类似,这些元件可整合到宿主细胞的染色体中,但其生命周期不涉及逆转录,且不具有传染性。与病毒方法相比,基于转座子的基因传递具有几个有利特征;然而,其效率一直是转座子应用的瓶颈。最近,我们采用一种新的体外进化策略,创建了脊椎动物特异性睡美人(SB)转座酶的一个新的高活性版本(SB100X)。当SB100X与增强型反向末端重复结构T2型SB转座子结合时,在哺乳动物细胞中的活性比原型高100多倍。我们建立了SB100X介导的啮齿动物转基因方案,平均产生35%的转基因奠基者,每个奠基者的转基因插入平均数较低(1-2个)。由于这些特性,基于SB100X的方案为体内敲低实验设计基于SB的转基因开辟了可能性。同样,SB转座子系统引入的单拷贝转基因单元不仅不太容易发生转基因沉默,还能更好地控制转基因表达水平和模式。