TAT-phiC31 integrase mediates DNA recombination in mammalian cells.
TAT-phiC31 integrase mediates DNA recombination in mammalian cells.
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DOI：
10.1016/j.jbiotec.2009.03.018
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发表时间：
2009-06
影响因子：
4.1
通讯作者：
Mao-xiang Zhang;Zhi-hui Li;Yu-Xiang Fang;Huan-zhang Zhu;J. Xue;Jin-zhong Chen;W. Jia
中科院分区：
文献类型：
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作者：
Mao-xiang Zhang;Zhi-hui Li;Yu-Xiang Fang;Huan-zhang Zhu;J. Xue;Jin-zhong Chen;W. Jia
链霉菌噬菌体整合酶phiC 31能够介导哺乳动物基因组中的位点特异性插入。为了避免phiC 31在宿主细胞中长期表达的潜在毒性，我们开发了一种采用细胞可渗透的TAT-phiC 31整合酶的方法。产生并纯化具有或不具有HIV达特细胞间转导肽的His 6标记的phiC 31蛋白。它们在体外均保持整合酶活性。然而，TAT-phiC 31而不是phiC 31能够介导293-PB [EGFP]报告细胞系基因组中两个att位点之间的特异性整合。转导的TAT-phiC 31主要定位在细胞质中，这与通过cDNA转染表达时phiC 31的定位相似。将核定位信号（NLS）肽添加到TAT-phiC 31的C-末端促进了整合酶的核定位，并提高了报告细胞系中重组的效率。这些结果表明，达特可以介导phiC 31蛋白进入细胞膜，从而在哺乳动物细胞中进行位点特异性整合。这是一种简单且可能更安全的用于基因递送的位点特异性重组方法。
Streptomyces phage integrase phiC31 is capable of mediating site-specific insertions in mammalian genomes. To avoid potential toxicity of long-term expression of phiC31 in host cells, we developed a method employing a cell-permeable TAT-phiC31 integrase. His6-tagged phiC31 proteins with or without an HIV TAT intercellular transducing peptide were generated and purified. Both of them retained integrase activity in vitro. However, TAT-phiC31 but not phiC31 was able to mediate a specific integration between two att sites in the genome of 293-PB [EGFP] report cell line. Transduced TAT-phiC31 was mainly localized in the cytoplasm that is similar to the localization of phiC31 when expressed through cDNA transfection. Adding a nuclear localization signal (NLS) peptide to the C-terminus of TAT-phiC31 facilitated nuclear localization of the integrase with an increased efficiency of recombination in the reporter cell line. These results demonstrated that TAT can mediate a cell membrane entry of phiC31 protein to perform a site-specific integration in mammalian cells. This is a simple and possibly safer method of site-specific recombination for gene delivery.