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

由于在胚胎发生过程中过量产生的基因产物具有毒性，系统地转基因表达感兴趣的基因有时会导致胚胎致死。为了研究这些基因，需要在出生后有条件地表达转基因。我们使用“TetOn系统”构建了系统表达催化前列腺素生物合成的COX-2和/或mPGES的条件转基因小鼠。在系统中，rtTA在强力霉素（DOX）处理下激活TRE调控基因的转录。因此，rtTA和TRE转基因小鼠都是诱导系统所必需的。首先，我们构建了几株trec - cox2和trec - cox -2/mPGES （trec - c2me）小鼠。为了检测rtTA和DOX对这些基因的诱导作用，将编码CMV-rtTA的adcmvrtta -腺病毒通过尾静脉感染转基因小鼠，并用DOX处理3-5天。在治疗小鼠的肝细胞中，证实了腺病毒rtTA的转导，并诱导了COX-2和mPGES。在几个品系中，TRE-COX-2和TRE-C2mE各有一个高诱导水平。Northern印迹分析证实了这些基因的诱导作用。接下来，我们构建了由普遍存在的启动子ROSA26驱动的表达rtTA的转基因小鼠。虽然在ROSA26-rtTA转基因小鼠的大多数组织中检测到rtTA的mRNA，但在肝细胞中腺病毒trel - lacz对rtTA和DOX没有反应。因此，rtTA基因在体内很可能是转录而非翻译的。研究表明，细菌rtTA密码子的使用频率与哺乳动物基因有很大不同。因此，我们构建了另一个表达密码子优化rtTA的转基因系（optirtTA）。虽然我们尚未在体内检测optirtTA的转录活性，但RQSA26-opti-rtTA小鼠有望构建COX-2和mPGES的系统性条件转基因小鼠。这些模型将为今后研究炎症、肿瘤的发展和转移提供有用的工具

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 … More 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