PiggyBac transposon-mediated gene transfer in human cells
PiggyBac transposon-mediated gene transfer in human cells
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DOI：
10.1038/sj.mt.6300028
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发表时间：
2007-01-01
影响因子：
12.4
通讯作者：
George, Alfred L., Jr.
中科院分区：
文献类型：
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作者：
Wilson, Matthew H.;Coates, Craig J.;George, Alfred L., Jr.
转座子是可用于将转基因整合到宿主细胞基因组中的移动的遗传元件。piggyBac转座子系统已用于昆虫的转基因和小鼠的种系诱变。我们比较了piggyBac与睡美人（SB）的转座活性，SB是一种广泛用于临床前基因治疗研究的转座子系统。具有最小长度50和30个末端重复的工程化piggyBac转座子在转染的培养的人细胞中表现出比充分表征的超活性SB系统更大的转座活性。PiggyBac切除是非常精确的，如在转座子切除位点典型地不存在“足迹”突变所证明的。我们在人类细胞中绘制了575个piggyBac整合位点，以确定基因组整合的位点选择性。PiggyBac表现出非随机整合位点的选择性，不同于以前报道的SB，包括更高的偏好，在转录起始位点周围的区域和长末端重复元件内的整合。重要的是，piggyBac没有观察到过度生产抑制，这是SB系统的主要限制。这允许产生组合“辅助-非依赖性”piggyBac转座酶-转座子载体，与用单独的转座子和转座酶质粒转染的细胞相比，其在人细胞中表现出2倍的转座活性增加。我们的结论是，piggyBac是一个转座子系统，具有一定的属性，包括高效率和缺乏过度生产的抑制，有利于转座子为基础的基因治疗的临床前发展。
Transposons are mobile genetic elements that can be used to integrate transgenes into host cell genomes. The piggyBac transposon system has been used for transgenesis of insects and for germline mutagenesis in mice. We compared transposition activity of piggyBac with Sleeping Beauty (SB), a widely used transposon system for preclinical gene therapy studies. An engineered piggyBac transposon with minimal length 50 and 30 terminal repeats exhibited greater transposition activity in transfected cultured human cells than a well-characterized hyperactive SB system. PiggyBac excision was very precise as evidenced by the typical absence of "footprint'' mutations at the site of transposon excision. We mapped 575 piggyBac integration sites in human cells to determine site selectivity of genomic integration. PiggyBac demonstrated non-random integration site selectivity that differed from that previously reported for SB, including a higher preference for integrations in regions surrounding transcriptional start sites and within long terminal repeat elements. Importantly, overproduction inhibition was not observed with piggyBac, a major limitation of the SB system. This permitted the generation of combination "helper-independent'' piggyBac transposase - transposon vectors that exhibited a 2-fold increase of transposition activity in human cells as compared with cells transfected with separate transposon and transposase plasmids. We conclude that piggyBac is a transposon system with certain properties, including high efficiency and lack of overproduction inhibition that are advantageous in preclinical development of transposon-based gene therapy.