Faculty of Biomedical and Life Sciences, University of Glasgow, Bower Building, University Ave, Glasgow G12 8QQ, Scotland, UK.
Nucleic Acids Res. 2010 Mar;38(4):1204-16. doi: 10.1093/nar/gkp1068. Epub 2009 Dec 3.
Transposons of the Tc1/mariner family have been used to integrate foreign DNA stably into the genome of a large variety of different cell types and organisms. Integration is at TA dinucleotides located essentially at random throughout the genome, potentially leading to insertional mutagenesis, inappropriate activation of nearby genes, or poor expression of the transgene. Here, we show that fusion of the zinc-finger DNA-binding domain of Zif268 to the C-terminus of ISY100 transposase leads to highly specific integration into TA dinucleotides positioned 6-17 bp to one side of a Zif268 binding site. We show that the specificity of targeting can be changed using Zif268 variants that bind to sequences from the HIV-1 promoter, and demonstrate a bacterial genetic screen that can be used to select for increased levels of targeted transposition. A TA dinucleotide flanked by two Zif268 binding sites was efficiently targeted by our transposase-Zif268 fusion, suggesting the possibility of designer 'Z-transposases' that could deliver transgenic cargoes to chosen genomic locations.
Tc1/mariner 家族的转座子已被用于将外源 DNA 稳定地整合到各种不同类型的细胞和生物体的基因组中。整合发生在 TA 二核苷酸上，位置基本随机分布在整个基因组中，可能导致插入突变、附近基因的不当激活或转基因表达不良。在这里，我们表明，Zif268 的锌指 DNA 结合域与 ISY100 转座酶的 C 末端融合，导致高度特异性地整合到 Zif268 结合位点一侧 6-17 个碱基对的 TA 二核苷酸上。我们表明，使用结合 HIV-1 启动子序列的 Zif268 变体可以改变靶向的特异性，并证明可以使用细菌遗传筛选来选择增加靶向转座的水平。由两个 Zif268 结合位点侧翼的 TA 二核苷酸被我们的转座酶-Zif268 融合物有效地靶向，这表明有可能设计出“Z-转座酶”，可以将转基因货物递送到选定的基因组位置。
