| Summary: | 碩士 === 國立陽明大學 === 生物醫學影像暨放射科學系暨研究所 === 98 === Objectives: Exogenous gene expression which can stimulate and manipulate by radiation excitation is current interest in gene strategies for cancer therapy. We expect that the radiation-responsive CArG element has been used to enhancer combine with the basal CMV promoter would result in ubiquitous expression in most of cells. This study, we show that the modulation of pure CArG element expression, using two-step transcriptional amplification (TSTA) system for image reporter gene expression.
Method: Start from synthetic CArG sequence was introduced into luciferase vector, we obtained nine directly repeated of CArG elements (denoted pE9-Fluc), eighteen (pE18-Fluc), and thirty -one (pE31-Fluc).Then we also constructed Enh CMV -E9-Fluc which linked cytomegalovirus (CMV) enhancer in front of E9, pE9nlsCRE contained the Cre recombinase protein, and pSTOPluc containing stop cassette link to luciferase reporter gene. And selected H1299 pSTOPluc stable clones were used for in vivo and in vitro study after luciferase assay validation.
Result: In luciferase assay, these “promoterless” CArG elements (E9, E18, and E31) showed showed transcription activation increase above1.5 folds after 2 Gy radiation exposures. The promoter transcriptional activity of Enh CMV -E9-Fluc was more than pE9-luc about 2 folds. And in the presence of 2 Gy IR triggering; the radiation enhancement still is retained as parental E9-FLuc construct present. Then we have validated methods to enhance the transcriptional activity by introducing a two-step transcriptional amplification (TSTA) system which contained pE9nlsCRE and pSTOPluc. Surprisingly under the IR exposure, the luciferase activity not only amplified but also the radiation mediated excitation still exists. Finally, the TSTA system was accomplished in vivo by using subcutaneous injection pSTOPluc stable clones, and transfected pE9nlsCRE in vivo to induce gene expression. The in vivo results were identical to the in vitro data that pE9nlsCRE was radiation inducible and manipulated the downstream luciferase gene expression.
Conclusion: The radio-responsive enhancer, containing ‘CArG’ elements was well known to be transiently activated by ionizing radiation. But we applied ‘CArG’ elements directly as a radio-active promoter not an enhancer; our finding intensely supports this hypothesis. We believe this type of molecular switch will be beneficial in spatial and temporal control exogenous gene expression.
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