Comparative Analysis of Transposable Element Vector Systems in Human Cells
Comparative Analysis of Transposable Element Vector Systems in Human Cells
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DOI：
10.1038/mt.2010.47
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发表时间：
2010-06-01
影响因子：
12.4
通讯作者：
Ivics, Zoltan
中科院分区：
文献类型：
--
作者：
Grabundzija, Ivana;Irgang, Markus;Ivics, Zoltan
转座子基因载体已成为脊椎动物遗传学中不可或缺的工具，其应用范围从模式物种的插入诱变和转基因到人类的基因治疗。转座子工具箱正在扩大，但一直缺乏一个仔细的，并排的不同转座子系统的特性。在这里，我们比较了睡美人（SB），piggyBac（PB）和Tol 2转座子的整体活性，过量生产抑制（OPI），靶位点选择，转基因拷贝数以及在人类细胞中的长期表达。在转座子DNA的可用性限制转座反应的条件下，SB是最有效的系统，包括难以转染的造血干/祖细胞（HSC），并且对OPI最敏感，这支持了对转座子组分进行仔细优化的需要。SB和PB的活性大致相同，并且在非限制性条件下比Tol 2更有效。所有三种系统都在人类细胞中提供了长期的转基因表达，沉默的迹象最小。事实上，Tol 2插入位点的定位揭示了H3 K27 me 3组蛋白标记的染色体区域内的显著代表性不足，该标记通常与转录抑制的异染色质相关。SB，Tol 2和PB构成了哺乳动物细胞中基因转移的互补研究工具，对基础和转化研究具有重要意义。
Transposon-based gene vectors have become indispensable tools in vertebrate genetics for applications ranging from insertional mutagenesis and transgenesis in model species to gene therapy in humans. The transposon tool-kit is expanding, but a careful, side-by-side characterization of the diverse transposon systems has been lacking. Here we compared the Sleeping Beauty (SB), piggyBac (PB), and Tol2 transposons with respect to overall activity, overproduction inhibition (OPI), target site selection, transgene copy number as well as long-term expression in human cells. SB was the most efficient system under conditions where the availability of the transposon DNA is limiting the transposition reaction including hard-to-transfect hematopoietic stem/progenitor cells (HSCs), and the most sensitive to OPI, underpinning the need for careful optimization of the transposon components. SB and PB were about equally active, and both more efficient than Tol2, under nonrestrictive conditions. All three systems provided long-term transgene expression in human cells with minimal signs of silencing. Indeed, mapping of Tol2 insertion sites revealed significant underrepresentation within chromosomal regions with H3K27me3 histone marks typically associated with transcriptionally repressed heterochromatin. SB, Tol2, and PB constitute complementary research tools for gene transfer in mammalian cells with important implications for fundamental and translational research.