Phage phiC31 integrase-mediated genomic integration and long-term gene expression in the lung after nonviral gene delivery
Phage phiC31 integrase-mediated genomic integration and long-term gene expression in the lung after nonviral gene delivery
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DOI：
10.1002/jgm.1090
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发表时间：
2007-11-01
影响因子：
3.5
通讯作者：
Rudolph, Carsten
中科院分区：
文献类型：
--
作者：
Aneja, Manish Kumar;Imker, Rabea;Rudolph, Carsten
背景 噬菌体 phi C31 整合酶已成为在不同组织中实现长期基因表达的有效工具。本研究调查了小鼠肺中phi C31整合酶的活性。方法用Lipofectamine 2000对小鼠肺泡上皮(MLE12)细胞进行转染。为了体内基因递送，将DNA与聚乙烯亚胺(PEI)复合，并将PEI-DNA复合物静脉注射到小鼠体内。通过体内生物发光成像监测小鼠体内荧光素酶的表达。通过聚合酶链式反应 (PCR) 证实了基因组整合和整合到先前描述的“热点”中。结果 phi C31 整合酶介导 MLE12 细胞中野生型 attB 和 attP 位点之间的分子内重组。在存在整合酶的情况下，可以在 MLE12 细胞中观察到长期基因表达，而无需任何选择压力。静脉注射PEI-DNA复合物后，荧光素酶的长期表达仅在与整合酶编码质粒共注射的小鼠肺中观察到。增加整合酶质粒的量和施用第二剂对单剂获得的荧光素酶表达水平没有影响，这比施用后“第一天”观察到的值低三个数量级。通过 PCR 证实了转基因在小鼠肺部的基因组整合。 15 只接受治疗的小鼠中有 7 只在 mpsL1 位点显示整合，这是之前描述的肝脏“热点”。 结论 这些结果为 phi C31 整合酶在肺中的活性提供了证据，但也强调需要优化系统以维持长期高水平的基因表达。版权所有（D 2007 John Wiley & Sons, Ltd.）
Background Phage phi C31 integrase has emerged as a potent tool for achieving long-term gene expression in different tissues. The present study investigated the activity of phi C31 integrase in murine lungs.Methods Transfections in murine alveolar epithelial (MLE12) cells were performed with Lipofectamine 2000. For in vivo gene delivery, DNA was complexed with polyethylenimine (PEI) and PEI-DNA complexes were injected intravenously into mice. Expression of luciferase in mice was monitored by in vivo bioluminsecence imaging. Genomic integration and integration into a previously described 'hotspot' were confirmed by polymerase chain reaction (PCR).Results phi C31 integrase mediated intramolecular recombination between wild-type attB and attP sites in MLE12 cells. Long-term gene expression could be observed in MLE12 cells in the presence of integrase without any selection pressure. Long-term expression of luciferase after intravenous injection of PEI-DNA complexes could be observed only in the lungs of mice which were co-injected with the integrase-encoding plasmid. Increased amounts of integrase plasmid and administration of a second dose had no effect on the level of luciferase expression achieved with a single dose, which was three orders of magnitude lower than the values observed on 'day 1' post application. Genomic integration of the transgene in the mouse lungs was confirmed by PCR. Seven out of the fifteen treated mice showed integration at the mpsL1 site, a previously described 'hot spot' from liver.Conclusions These results provide evidence for the activity of phi C31 integrase in lungs but also emphasize the need for optimization of the system to maintain long-term gene expression at high levels. Copyright (D 2007 John Wiley & Sons, Ltd.