| Project/Area Number |
18500374
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Medical systems
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| Research Institution | University of Toyama |
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Principal Investigator |
OGAWA Ryohei University of Toyama, Faculty of Medicine, Lecturer (60334736)
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| Co-Investigator(Kenkyū-buntansha) |
KONDO Takashi University of Toyama, Faculty of Medicine, Professor (40143937)
ZHAO Qing-Li University of Toyama, Faculty of MedicineF, Assistant professor (90313593)
LEE Sung-il Kansai Medical University, Institute of Biomedical Science, Associate Professor (90361964)
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| Project Period (FY) |
2006 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥3,650,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥450,000)
Fiscal Year 2007: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
Fiscal Year 2006: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Keywords | Promoter / cis-element / TATA box / error prone PCR / ultrasound |
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| Research Abstract |
An artificial promoter is suitable for gene therapy vectors since it could be easily integrated with a novel property. In particular, a responsive promoter would be useful in various field of gene therapy and vectors with such promoters are considered safer and more efficacious. Promoters are composed of a sequence including the TATA box to which basic transcription factors bind and cis-elements to which active transcription factors bind. We have attempted to construct artificial promoters of interest by constructing DNA fragments of randomly combining cis-elements appropriately selected for a purpose and attaching it to the TATA box.
Activity of the strongest promoter of 11 artificial promoters that were constructed using cis-elements of NF-kB, NF-Y, AP-1 and CBF-A, transcription factors that are active in various tissues, was almost equivalent to those of SV40 promoters and CMV promoters. In addition, the most responsive promoter to X-rays out of the 11 promoters was improved through
… More
introduction of random point mutations by error prone PCR to cause 20-fold increase in gene expression in response to 10 Gy X-ray irradiation.
It is known that ultrasound irradiation could induce intracellular oxidative stress that would be a similar intracellular environment caused by X-ray irradiation. We thus introduce the artificial promoter responsive to X-ray into cells and irradiate the cells with 1 MHz ultrasound at 1 W/cm^2 to see if ultrasound irradiation might cause promoter activation, showing 2-fold increase in gene expression. In addition, the improved one with point mutations showed 6-fold increase in gene expression in response to the ultrasound irradiation. The improved promoter was then stably introduced into the genome of a cell to establish cell lines. Such a cell line was subcutaneously transplanted on to mice. Although it was exposed to ultrasound irradiation after tumor formation, significant increase in gene expression was not observed. We therefore obtained 2 more sensitive promoters to ultrasound irradiation showing more than 10-fold increase in gene expression out of 62 artificial promoter clones constructed similarly to the 11 artificial promoters. One of the two was improved with point mutation introduction and showed more than 20-fold increase in gene expression. We are going to examine activation of the promoter in vivo and its effect on the expression control of therapeutic genes. Less
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