School of Allied Health Sciences, Kitasato University, 1-15-1 Sagamihara, Kanagawa 228-8555, Japan.
J Biosci Bioeng. 2010 Jul;110(1):118-23. doi: 10.1016/j.jbiosc.2009.12.013. Epub 2010 Feb 4.
A promoter that augments gene expression in response to stimulation of ionizing radiation would be a desired tool for radiogenetic therapy, a combination of radiotherapy and gene therapy. Although various promoters occurring naturally or artificially have been used for researches, one showing higher reactivity to ionizing radiation is desirable. In the present study, we attempted to improve a radiation-responsive promoter of the p21 through a technique called DNA shuffling. A library of DNA fragments was constructed by re-ligation of randomly digested promoter fragments and improved promoters were chosen out of the library. We repeated this process twice to obtain a promoter showing 2.6-fold better reactivity to ionizing radiation compared with its parent, p21 promoter after 10 Gy gamma-ray irradiation. Nucleotide sequence analyses revealed that the obtained promoter was densely packed with some of the cis-acting elements including binding sites for p53, NF-kappaB, NRF-2, AP-1 and NF-Y more than p21 promoter. In addition, it was shown that its induction by ionizing radiation was dependent upon p53 status of a cell line, suggesting that the promoter retained properties of the p21 promoter. This technique is simple and efficient to improve a promoter responsive to other stimulus of interest besides IR.
一种能在受到电离辐射刺激时增强基因表达的启动子，将成为放射基因治疗（放疗与基因治疗的结合）的理想工具。尽管已经使用了各种天然或人工的启动子进行研究，但人们还是希望能有一种对电离辐射有更高反应性的启动子。在本研究中，我们试图通过一种称为 DNA 重排的技术来改进 p21 的辐射响应启动子。通过随机消化启动子片段并重新连接，构建了一个 DNA 片段文库，然后从文库中选择出改进后的启动子。我们重复这个过程两次，获得了一个在受到 10Gy 伽马射线照射后，比其亲本 p21 启动子对电离辐射反应性提高了 2.6 倍的启动子。核苷酸序列分析表明，与 p21 启动子相比，获得的启动子含有更多的顺式作用元件，包括 p53、NF-κB、NRF-2、AP-1 和 NF-Y 的结合位点。此外，还表明其对电离辐射的诱导依赖于细胞系中 p53 的状态，这表明该启动子保留了 p21 启动子的特性。除了电离辐射之外，这种技术对于改进对其他感兴趣的刺激有反应的启动子也很简单有效。
