Site-directed transposon integration in human cells.
Site-directed transposon integration in human cells.
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DOI：
10.1093/nar/gkm089
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发表时间：
2007
影响因子：
14.9
通讯作者：
Kay MA
中科院分区：
文献类型：
--
作者：
Yant SR;Huang Y;Akache B;Kay MA
睡美人转座子是一种很有前途的基因转移载体，它可以非特异性地整合到宿主细胞基因组中。在此，我们试图通过将位点特异性DNA结合结构域（DBD）偶联到SB转座酶来指导转座子整合到预定的DNA位点中。我们设计了融合蛋白，该融合蛋白由通过可变长度接头连接到多指锌指蛋白E2 C两端的超活性SB转座酶（HSB 5）组成，该蛋白结合人类17号染色体上的独特序列。虽然DBD连接到SB的C-末端消除了人细胞转座测定中的活性，但N-末端添加E2 C或Gal 4 DBD没有。分子分析表明，这些DBD-SB融合蛋白保留了其各自的底物分子的DNA结合特异性，并能够介导真正的转座反应。我们还表征了E2 C-SB融合蛋白存在下的转座子整合，以确定其靶向预定义DNA位点的潜力。我们的研究结果表明，融合蛋白介导的拴系可以有效地重定向转座子插入位点的选择在人类细胞中，但建议整合复合物的稳定对接也可能部分干扰剪切和粘贴机制。这些发现说明了定向转座子整合的可行性，并强调了未来发展的潜在手段。
The Sleeping Beauty (SB) transposon is a promising gene transfer vector that integrates nonspecifically into host cell genomes. Herein, we attempt to direct transposon integration into predetermined DNA sites by coupling a site-specific DNA-binding domain (DBD) to the SB transposase. We engineered fusion proteins comprised of a hyperactive SB transposase (HSB5) joined via a variable-length linker to either end of the polydactyl zinc-finger protein E2C, which binds a unique sequence on human chromosome 17. Although DBD linkage to the C-terminus of SB abolished activity in a human cell transposition assay, the N-terminal addition of the E2C or Gal4 DBD did not. Molecular analyses indicated that these DBD-SB fusion proteins retained DNA-binding specificity for their respective substrate molecules and were capable of mediating bona fide transposition reactions. We also characterized transposon integrations in the presence of the E2C-SB fusion protein to determine its potential to target predefined DNA sites. Our results indicate that fusion protein-mediated tethering can effectively redirect transposon insertion site selection in human cells, but suggest that stable docking of integration complexes may also partially interfere with the cut-and-paste mechanism. These findings illustrate the feasibility of directed transposon integration and highlight potential means for future development.
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影响因子：
4.8
作者：
Cherepanov, P;Maertens, G;Debyser, Z
通讯作者：
Debyser, Z
影响因子：
9.2
作者：
Haas, J;Park, EC;Seed, B
通讯作者：
Seed, B
影响因子：
158.5
作者：
Hacein-Bey-Abina, S;Le Deist, F;Leiva, L
通讯作者：
Leiva, L
DOI：
10.1073/pnas.91.20.9233
发表时间：
1994-09-27
影响因子：
11.1
作者：
BUSHMAN, FD
通讯作者：
BUSHMAN, FD
影响因子：
4.8
作者：
Cherepanov, P;Pluymers, W;Debyser, Z
通讯作者：
Debyser, Z