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Construction of X‐ray‐inducible promoters through cis‐acting element elongation and error‐prone polymerase chain reaction
Author(s) -
Ogawa Ryohei,
Lee Sungil,
Kagiya Go,
Hirano Hisao,
Fukuda Shigekazu,
Kondo Takashi,
Kodaki Tsutomu
Publication year - 2008
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.1154
Subject(s) - promoter , luciferase , tata box , microbiology and biotechnology , clone (java method) , transcription (linguistics) , biology , gene , caat box , transfection , gene expression , genetics , linguistics , philosophy
Background A 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 methods Binding 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). Results Of 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. Conclusion A 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.