2002 Fiscal Year Final Research Report Summary
時期特異的な全身性遺伝子発現誘導トランスジェニックマウスシステムの開発
| Project/Area Number |
13680912
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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 |
Laboratory animal science
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
OSHIMA Masanobu Kyoto University, Graduate School of Medicine, Associate Professor, 医学研究科, 助教授 (40324610)
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| Co-Investigator(Kenkyū-buntansha) |
ISHIKAWA Tomoo Kyoto University, Graduate School of Medicine, Assistant Professor, 医学研究科, 助手 (70322162)
TAKETO Makoto Kyoto University, Graduate School of Medicine, Professor, 医学研究科, 教授 (70281714)
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| Project Period (FY) |
2001 – 2002
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| Keywords | transgenic mice / conditional / tetracycline / doxycycline / COX-2 Transeenics / rtTA / TRE / opti-rtTA |
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| Research Abstract |
Systemic transgenic expression of the gene of interest occasionally results in embryonic lethal because of toxicity of the over-produced gene product during embryogenesis. Conditional expression of the transgene after birth is required for investigation of such genes. We used "TetOn system" for construction of conditional transgenic mice systemically expressing COX-2 and/or mPGES that catalizes prostaglandin biosynthesis. In the system, rtTA activates transcription of TRE regulated genes under doxycycline (DOX) treatment. Accordingly, both rtTA and TRE transgenic mice are necessary for the induction system. First, we constructed several lines of TRE-COX2 and TRE-COX-2/mPGES (TRE-C2mE) mice. To examine the induction of these genes by rtTA and DOX, transgenic mice were infected through tail vein with AdCMVrtTA-adenovirus encoding CMV-rtTA and treated with DOX for 3-5 days. In the hepatocytes of the treated mice, adenoviral rtTA transduction was confirmed and induction of COX-2 and mPGES
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was detected. Among several lines, one for each TRE-COX-2 and TRE-C2mE showed high level of induction. Induction of these genes was confirmed by Northern blotting analysis. We next constructed transgenic mice expressing rtTA driven by ROSA26 promoter, a ubiquitous promoter. Although mRNA for rtTA was detected in most tissues of ROSA26-rtTA transgenic mice, adenoviral TRE-LacZ did not respond to rtTA and DOX in hepatocytes. Accordingly, it is likely that rtTA gene is transcribed but not translated in vivo. It has been shown that frequency of codon usage of bacterial rtTA is quite different from mammalian genes. Thus, we constructed another transgenic line expressing codonoptimized rtTA (optirtTA). Although we have not examined the transcriptional activity of optirtTA in vivo yet, RQSA26-opti-rtTA mice are expected to construct the systemic conditional transgenic mice for COX-2 and mPGES. These models will be useful tool for future investigation of inflammation, cancer development and metastasis Less
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