Gene expression promoted by the SV40 DNA targeting sequence and the hypoxia-responsive element under normoxia and hypoxia
Gene expression promoted by the SV40 DNA targeting sequence and the hypoxia-responsive element under normoxia and hypoxia
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DOI：
10.1590/s0100-879x2010000800005
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发表时间：
2010-08-01
影响因子：
2.3
通讯作者：
Han, S.W.
中科院分区：
文献类型：
--
作者：
Sacramento, C.B.;Moraes, J.Z.;Han, S.W.
本研究的主要目的是寻找合适的DNA靶向序列（DNA-targeting sequences，RT-PCR）构建质粒载体，用于治疗缺血性疾病。利用已知的猴病毒40核转录因子（SV 40-HRE）和缺氧反应元件（HRE）序列构建表达人血管内皮生长因子（hVEGF）基因的质粒载体。测定了常氧（20%O-2）和缺氧（低于5%O-2）条件下质粒核转运率和随之而来的基因表达。构建含有SV 40-HRE或HRE序列的质粒，并用于体外转染A293 T细胞系（人胚肾细胞系）和体内转染小鼠骨骼肌细胞。通过实时PCR监测质粒向细胞核的转运，并通过ELISA测量hVEGF基因的表达水平。SV 40-HRE质粒的体外核转运效率在缺氧条件下降低约50%，而HRE质粒在缺氧条件下提高约50%。在体外和体内，在缺氧和常氧条件下，报告基因表达的定量，证实了SV 40-CMV质粒在常氧条件下的功能更好，而HRE质粒在缺氧条件下是上级。这些结果表明，含有DNA结合序列的质粒的基因表达效率受到培养基中氧浓度的影响。
The main objective of the present study was to find suitable DNA-targeting sequences (DTS) for the construction of plasmid vectors to be used to treat ischemic diseases. The well-known Simian virus 40 nuclear DTS (SV40-DTS) and hypoxia-responsive element (HRE) sequences were used to construct plasmid vectors to express the human vascular endothelial growth factor gene (hVEGF). The rate of plasmid nuclear transport and consequent gene expression under normoxia (20% O-2) and hypoxia (less than 5% O-2) were determined. Plasmids containing the SV40-DTS or HRE sequences were constructed and used to transfect the A293T cell line (a human embryonic kidney cell line) in vitro and mouse skeletal muscle cells in vivo. Plasmid transport to the nucleus was monitored by real-time PCR, and the expression level of the hVEGF gene was measured by ELISA. The in vitro nuclear transport efficiency of the SV40-DTS plasmid was about 50% lower under hypoxia, while the HRE plasmid was about 50% higher under hypoxia. Quantitation of reporter gene expression in vitro and in vivo, under hypoxia and normoxia, confirmed that the SV40-DTS plasmid functioned better under normoxia, while the HRE plasmid was superior under hypoxia. These results indicate that the efficiency of gene expression by plasmids containing DNA binding sequences is affected by the concentration of oxygen in the medium.