Optimization of Streptomyces bacteriophage φC31 integrase system to prevent post integrative gene silencing in pulmonary type II cells
Optimization of Streptomyces bacteriophage φC31 integrase system to prevent post integrative gene silencing in pulmonary type II cells
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DOI：
10.3858/emm.2009.41.12.098
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发表时间：
2009-12-31
影响因子：
12.8
通讯作者：
Rudolph, Carsten
中科院分区：
文献类型：
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作者：
Aneja, Manish Kumar;Geiger, Johannes;Rudolph, Carsten
PHI C31整合酶已成为实现基因在不同组织中长期表达的有力工具。本研究旨在优化肺泡II型细胞pHC31整合酶系统的组成要素。检测转染后不同时间点的荧光素酶和β-半乳糖苷酶活性。5-氮杂-2‘-脱氧胞苷(AZA)和曲古菌素A(TSA)分别抑制DNA甲基转移酶和组蛋白脱乙酰酶复合体(HDAC)。在A549细胞中，使用CMV启动子表达整合酶可获得最高的整合酶活性，而在MLE12细胞中，CAG和CMV启动子的作用相同。只在A549细胞中观察到PolA位点的作用，而用牛生长激素(BGH)PolyA位点取代SV40 PolyA会导致整合酶活性的增强。添加C端SV40核定位信号(NLS)并没有导致整合酶活性的显著增加。AZA和TSA的长期表达研究为整合后基因沉默提供了证据。在MLE12细胞中，DNA甲基酶和HDAC在沉默中起重要作用，而在A549细胞中，沉默主要归因于HDAC活性。在改进的骨架中含有细胞启动子泛素B(UBB)、泛素C(UCC)和延伸因子1α(EF1α)的供体质粒可防止整合后的基因沉默。与A549和MLE12细胞相比，在人支气管上皮细胞BEAS-2B中没有观察到沉默。在EF1α启动子下编码小鼠促红细胞生成素的供体质粒与Phi C31整合酶结合时，在静脉注射到肺部后，小鼠的长期促红细胞生成素表达和随后的红细胞压积水平都较高。这些结果为Phi C31整合酶后细胞特异性整合基因沉默提供了证据，并证明了供体质粒在该系统获得长期表达中的关键作用。
phi C31 integrase has emerged as a potent tool for achieving long-term gene expression in different tissues. The present study aimed at optimizing elements of phi C31 integrase system for alveolar type II cells. Luciferase and P-galactosidase activities were measured at different time points post transfection. 5-Aza-2'deoxycytidine (AZA) and trichostatin A (TSA) were used to inhibit DNA methyltransferase and histone deacetylase complex (HDAC) respectively. In A549 cells, expression of the integrase using a CMV promoter resulted in highest integrase activity, whereas in MLE12 cells, both CAG and CMV promoter were equally effective. Effect of polyA site was observed only in A549 cells, where replacement of SV40 polyA by bovine growth hormone (BGH) polyA site resulted in an enhancement of integrase activity. Addition of a C-terminal SV40 nuclear localization signal (NLS) did not result in any significant increase in integrase activity. Long-term expression studies with AZA and TSA, provided evidence for post-integrative gene silencing. In MLE12 cells, both DNA methylases and HDACs played a significant role in silencing, whereas in A549 cells, it could be attributed majorly to HDAC activity. Donor plasmids comprising cellular promoters ubiquitin B (UBB), ubiquitin C (UCC) and elongation factor 1 alpha (EF1 alpha) in an improved backbone prevented post-integrative gene silencing. In contrast to A549 and MLE12 cells, no silencing could be observed in human bronchial epithelial cells, BEAS-2B. Donor plasmid coding for murine erythropoietin under the EF1 alpha promoter when combined with phi C31 integrase resulted in higher long-term erythropoietin expression and subsequently higher hematocrit levels in mice after intravenous delivery to the lungs. These results provide evidence for cell specific post integrative gene silencing with phi C31 integrase and demonstrate the pivotal role of donor plasmid in long-term expression attained with this system.