Transcriptional analysis in vivo of the hepatic genes, Cyp2b9 and Cyp2b10, by intravenous administration of plasmid DNA in mice.
Transcriptional analysis in vivo of the hepatic genes, Cyp2b9 and Cyp2b10, by intravenous administration of plasmid DNA in mice.
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通过向小鼠静脉注射质粒 DNA，对肝基因 Cyp2b9 和 Cyp2b10 进行体内转录分析。
DOI：
10.1016/s0304-4165(02)00484-1
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
Kemper,Byron
中科院分区：
文献类型：
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作者：
Rivera-Rivera,Ilia;Kim,Jongsook;Kemper,Byron
CYP2B 基因的苯巴比妥 (PB) 反应性已被证明是由 PB 反应单位 (PBRU) 介导的。 PBRU 的核心包含两个核受体位点 NR-1 和 NR-2，以及一个核因子 1 (NF 1) 结合位点，这是 PB 反应所必需的，但核心侧翼序列的重要性尚不清楚。我们通过在小鼠尾静脉中静脉注射质粒 DNA 来转染体内肝细胞并分析 PB 诱导的序列要求。在此测定中，PB 处理使 Cyp2b10 PBRU 的反式激活增加约 100 倍，这与内源基因表达的增加相似，而 Cyp2b9 PBRU 无反应。对两个 PBRU 的嵌合体和 Cyp2b10 PBRU 的缺失突变体的分析表明，包含 NR-1、NR-2 和 NF-1 核心位点的核心区域不足以实现 PB 响应。核心序列 3' 侧的附加序列包括先前定义的辅助因子 1 (AF-1) 位点，部分恢复了响应性。该区域仅在 Cyp2b10 中包含 NF-1 的结合位点，而在 Cyp2b9 中则不包含，但 PB 响应性不需要完整的位点。纯化的组成型雄甾烷受体 (CAR)/类视黄醇 X 受体 (RXR) 与 Cyp2b10 中核心 NR-1 和 NR-2 位点以及核心 5' 侧的第三个 NR-3 位点结合。没有观察到 CAR/RXR 与 Cyp2b9 PBRU 的结合。这些结果表明，消除 CAR/RXR 结合的 NR 位点的变化足以导致 Cyp2b9 对 PB 无响应，但核心侧翼序列的变化独立地消除了 PB 响应。结果证明了通过尾静脉注射 DNA 体内转染小鼠肝细胞作为体内基因转录分析方法的优势，并表明 PBRU 核心区侧翼的序列是体内 PB 诱导所必需的。
Phenobarbital (PB) responsiveness of CYP2B genes has been shown to be mediated by a PB responsive unit (PBRU). The core of the PBRU contains two nuclear receptor sites, NR-1 and NR-2, and a nuclear factor-1 (NF 1) binding site, which are required for PB responsiveness, but the importance of sequences flanking the core is not clear. We have used intravenous administration of plasmid DNA in the tail veins of mice to transfect hepatocytes in vivo and analyze sequence requirements for PB induction. In this assay PB treatment increased transactivation by the Cyp2b10 PBRU about 100-fold, which is similar to the increase in the expression of the endogenous gene while the Cyp2b9 PBRU was unresponsive. Analysis of chimeras of the two PBRUs and deletion mutants of the Cyp2b10 PBRU indicated that the core region containing the NR-1, NR-2 and NF-1 core sites is not sufficient for PB responsiveness. Additional sequence at the 3′ side of the core sequence, which included a previously defined accessory factor-1 (AF-1) site, partially restored responsiveness. This region contained a binding site for NF-1 only in Cyp2b10 and not in Cyp2b9, but the intact site was not required for PB responsiveness. Purified constitutive androstane receptor (CAR)/retinoid X receptor (RXR) bound to the core NR-1 and NR-2 sites and to a third NR-3 site to the 5′ side of the core in Cyp2b10. No binding of CAR/RXR to the Cyp2b9 PBRU was observed. These results indicate that changes in the NR sites which eliminate CAR/RXR binding are sufficient for the non-responsiveness to PB of Cyp2b9, but changes in sequences flanking the core independently eliminate PB responsiveness. The results demonstrate the advantages of transfection of mouse hepatocytes in vivo by tail vein injection of DNA as a method for transcriptional analysis of genes in vivo and show that sequences flanking the core region of the PBRU are required for PB induction in vivo.