Gene transfer into genomes of human cells by the sleeping beauty transposon system
Gene transfer into genomes of human cells by the sleeping beauty transposon system
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DOI：
10.1016/s1525-0016(03)00099-6
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发表时间：
2003-07-01
影响因子：
12.4
通讯作者：
Hackett, PB
中科院分区：
文献类型：
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作者：
Geurts, AM;Yang, Y;Hackett, PB
睡美人（SB）转座子系统，来自硬骨鱼序列，是非常有效地将DNA传递到脊椎动物的基因组，包括人类。我们已经检查了SB系统的几个参数，以改善它作为一个潜在的，非病毒载体的基因治疗。我们的调查集中在三个功能：有效整合到HeLa细胞的染色体的转座子的承载能力，SB转座酶基因的过表达对转座率的影响，和SB转座酶的活性提高转基因插入细胞染色体的插入率的改善。我们发现约6 kb的SB转座子保留了50%的最大转座效率，这足以递送70-80%的具有适当转录调控序列的鉴定的人cDNA。过表达抑制研究表明，SB转座酶与转座子的最佳比例可获得最大的转座率，这表明两部分转座子系统的递送条件对于最佳的基因转移效率是重要的。我们进一步改进了SB转座酶，使其包含几个氨基酸取代，其结果是产生了一种称为SB 11的改进转座酶。使用SB 11，我们能够实现的转座率比通过随机重组插入染色体的质粒所实现的转座率高出约100倍。随着最近描述的转座子本身的改进，SB系统似乎是人类基因治疗的潜在基因转移工具。
The Sleeping Beauty (SB) transposon system, derived from teleost fish sequences, is extremely effective at delivering DNA to vertebrate genomes, including those of humans. We have examined several parameters of the SB system to improve it as a potential, nonviral vector for gene therapy. Our investigation centered on three features: the carrying capacity of the transposon for efficient integration into chromosomes of HeLa cells, the effects of overexpression of the SB transposase gene on transposition rates, and improvements in the activity of SB transposase to increase insertion rates of transgenes into cellular chromosomes. We found that SB transposons of about 6 kb retained 50% of the maximal efficiency of transposition, which is sufficient to deliver 70-80% of identified human cDNAs with appropriate transcriptional regulatory sequences. Overexpression inhibition studies revealed that there are optimal ratios of SB transposase to transposon for maximal rates of transposition, suggesting that conditions of delivery of the two-part transposon system are important for the best gene-transfer efficiencies. We further refined the SB transposase to incorporate several amino acid substitutions, the result of which led to an improved transposase called SB11. With SB11 we are able to achieve transposition rates that are about 100-fold above those achieved with plasmids that insert into chromosomes by random recombination. With the recently described improvements to the transposon itself, the SB system appears to be a potential gene-transfer tool for human gene therapy.