A Hyperactive Transposase Promotes Persistent Gene Transfer of a piggyBac DNA Transposon.
A Hyperactive Transposase Promotes Persistent Gene Transfer of a piggyBac DNA Transposon.
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多动性转座酶促进Piggybac DNA转座子的持续基因转移。
DOI：
10.1038/mtna.2012.12
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发表时间：
2012-10-16
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影响因子：
--
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非病毒载体系统越来越多地用于基因靶向和基因转移应用。piggyBac转座子代表了用于体内基因转移的替代整合载体。我们假设，该系统可以实现持久的基因转移到肝脏时，全身给药。我们报告说，一种新型的高度活跃的转座酶在人肝细胞系中产生了比密码子优化的转座酶更高的转座效率。在为期6个月的研究中，小鼠肝脏中高度活跃的转座酶介导的报告基因表达持续保持在密码子优化转座酶的两倍水平。值得注意的是，在部分肝切除术后的小鼠中表达持续存在，这与来自整合的转基因的表达一致。我们还利用高活性转座酶将人α1-抗胰蛋白酶基因导入小鼠体内，并在血清中获得了稳定表达。为了确定插入的整合模式，我们在人类细胞中进行了大规模作图，并回收了60，685个独特的过度活跃的转座酶介导的插入。我们发现，一个过度活跃的piggyBac转座酶赋予的整合模式改变，从昆虫piggyBac转座酶，与转录起始位点附近的整合频率比以前报道的降低。我们的研究结果支持piggyBac转座子结合的超活性转座酶是一个有效的整合载体系统，在体外和体内的应用。
Nonviral vector systems are used increasingly in gene targeting and gene transfer applications. The piggyBac transposon represents an alternative integrating vector for in vivo gene transfer. We hypothesized that this system could achieve persistent gene transfer to the liver when administered systemically. We report that a novel hyperactive transposase generated higher transposition efficiency than a codon-optimized transposase in a human liver cell line. Hyperactive transposase-mediated reporter gene expression persisted at levels twice that of codon-optimized transposase in the livers of mice for the 6-month study. Of note, expression persisted in mice following partial hepatectomy, consistent with expression from an integrated transgene. We also used the hyperactive transposase to deliver the human α1-antitrypsin gene and achieved stable expression in serum. To determine the integration pattern of insertions, we performed large-scale mapping in human cells and recovered 60,685 unique hyperactive transposase-mediated insertions. We found that a hyperactive piggyBac transposase conferred an altered pattern of integration from that of insect piggyBac transposase, with a decreased frequency of integration near transcription start sites than previously reported. Our results support that the piggyBac transposon combined with the hyperactive transposase is an efficient integrating vector system for in vitro and in vivo applications.
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