A hyperactive piggyBac transposase for mammalian applications
A hyperactive piggyBac transposase for mammalian applications
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DOI：
10.1073/pnas.1008322108
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发表时间：
2011-01-25
影响因子：
11.1
通讯作者：
Craig, Nancy L.
中科院分区：
文献类型：
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作者：
Yusa, Kosuke;Zhou, Liqin;Craig, Nancy L.
DNA转座子已广泛应用于各种生物的转基因和插入突变。在哺乳动物细胞中活跃的转座子中，蛾源转座子piggyBac以其高效的转座、大的货物容量和精确的供体位点修复而最有前途。在这里，我们报告了一个过度活跃的piggyBac转座酶的产生。piggyBac在多种生物体中的主动转座使我们能够在酵母中筛选转座酶突变体库，以寻找过度活跃的突变体，然后在小鼠ES细胞中测试候选物。我们在酵母中分离出18个高活性突变体，其中5个在哺乳动物细胞中也是高活性的。通过将所有突变（总共7个氨基酸取代）组合到单个阅读框中，我们产生了一种独特的高度活跃的piggyBac转座酶，其切除和整合分别增加了17倍和9倍。我们通过证明无转基因小鼠诱导多能干细胞的生成效率增加来展示其适用性。我们还分析了这种过度活跃的piggyBac转座酶是否影响宿主细胞的基因组完整性。由过度活跃的piggyBac转座酶留下的足迹的频率与WT转座酶一样低（类似于1%），并且我们没有发现转座酶的表达影响基因组完整性的证据。这种过度活跃的piggyBac转座酶扩展了piggyBac转座子在哺乳动物遗传学和基因治疗中的应用。
DNA transposons have been widely used for transgenesis and insertional mutagenesis in various organisms. Among the transposons active in mammalian cells, the moth-derived transposon piggyBac is most promising with its highly efficient transposition, large cargo capacity, and precise repair of the donor site. Here we report the generation of a hyperactive piggyBac transposase. The active transposition of piggyBac in multiple organisms allowed us to screen a transposase mutant library in yeast for hyperactive mutants and then to test candidates in mouse ES cells. We isolated 18 hyperactive mutants in yeast, among which five were also hyperactive in mammalian cells. By combining all mutations, a total of 7 aa substitutions, into a single reading frame, we generated a unique hyperactive piggyBac transposase with 17-fold and ninefold increases in excision and integration, respectively. We showed its applicability by demonstrating an increased efficiency of generation of transgene-free mouse induced pluripotent stem cells. We also analyzed whether this hyperactive piggyBac transposase affects the genomic integrity of the host cells. The frequency of footprints left by the hyperactive piggyBac transposase was as low as WT transposase (similar to 1%) and we found no evidence that the expression of the transposase affects genomic integrity. This hyperactive piggyBac transposase expands the utility of the piggyBac transposon for applications in mammalian genetics and gene therapy.