Construction of X‐ray‐inducible promoters through cis‐acting element elongation and error‐prone polymerase chain reaction

Journal of Gene Medicine - Tập 10 Số 3 - Trang 316-324 - 2008
Ryohei Ogawa1,2, Sung‐il Lee3,4, Go Kagiya2,5, Hisao Hirano6, Satoshi Fukuda2, Takashi Kondo1, Tsutomu Kodaki7
1Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194, Japan
2Medical Division, The Wakasa Wan Energy Research Center, Tsuruga 914-0192, Japan
3Division of Animal Resources and Development, Life Science Research Center, University of Toyama, Toyama, 930-0194, Japan
4Institute of Biomedical Science, Kansai Medical University, Moriguchi 570-8506, Japan
5Radiation Biology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
6Research Division, Nippon Gene Co., Ltd., Toyama 930-0834, Japan
7Bioenergy Research Section, Institute of Advanced Energy, Kyoto University, Uji 611-0011, Japan

Tóm tắt

AbstractBackgroundA promoter that is activated by ionizing radiation may be a useful tool for cancer therapy since, with such a promoter, the therapeutic gene can be expressed only in cancer tissues by irradiation. An artificially constructed promoter is advantageous as natural promoters may have physiological limitations. However, reasonably designing a promoter is hampered by shortage of information about the relationship between the structure and properties of a promoter DNA.Materials and methodsBinding sites of four transcription factors that were activated by radiation were randomly ligated and linked to a TATA‐box sequence to control the luciferase gene located downstream. Transiently transfected cancer cells with such a vector were exposed to X‐ray irradiation and enhancement of luciferase expression was assessed. To improve promoter sensitivity, mutations were randomly introduced into a constructed promoter by error‐prone polymerase chain reaction (epPCR).ResultsOf the 11 promoters constructed, the clone 11 promoter (clone 11 + TATA‐box) showed a 5‐fold enhancement 6 h after the 10 Gy X‐ray irradiation in HeLa cells. A mutant designated the clone 11‐9‐37 promoter generated through two steps of epPCR showed a sensitivity 4.8 times higher than the clone 11 promoter to the 10 Gy X‐rays, showing 21.6‐fold enhancement of luciferase expression. Clone 11 was composed of 16 cis‐acting elements, and the clone 11‐9‐37 promoter carried six point mutations.ConclusionA sensitively responsive promoter to radiation could be constructed using this method, possibly leading to the construction of a promoter of interest that could be applied for clinical use. Copyright © 2007 John Wiley & Sons, Ltd.

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Journal of Gene Medicine

Tập 10 Số 3

316-324

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