Yant Stephen R, Huang Yong, Akache Bassel, Kay Mark A
Department of Pediatrics and Genetics, Stanford University School of Medicine, Stanford, CA 94305-5208, USA.
Nucleic Acids Res. 2007;35(7):e50. doi: 10.1093/nar/gkm089. Epub 2007 Mar 7.
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.
睡美人（SB）转座子是一种很有前景的基因转移载体，可非特异性地整合到宿主细胞基因组中。在此，我们尝试通过将位点特异性DNA结合结构域（DBD）与SB转座酶偶联，将转座子整合定向到预定的DNA位点。我们构建了融合蛋白，该融合蛋白由一个高活性SB转座酶（HSB5）通过可变长度接头连接到多锌指蛋白E2C的两端组成，E2C可与人17号染色体上的独特序列结合。尽管在人细胞转座试验中，DBD与SB的C末端连接会消除活性，但在SB的N末端添加E2C或Gal4 DBD则不会。分子分析表明，这些DBD-SB融合蛋白对各自的底物分子保留了DNA结合特异性，并且能够介导真正的转座反应。我们还对存在E2C-SB融合蛋白时的转座子整合进行了表征，以确定其靶向预定义DNA位点的潜力。我们的结果表明，融合蛋白介导的拴系可有效重定向人细胞中转座子插入位点的选择，但表明整合复合物的稳定对接也可能部分干扰剪切粘贴机制。这些发现说明了定向转座子整合的可行性，并突出了未来发展的潜在手段。
