The current Xenopus ORFeome contains ~10,250 validated, full‐length cDNA sequences without stop codons from Xenopus laevis and ~3,970 from Xenopus tropicalis cloned into Gateway‐compatible entry vectors. To increase the utility of the ORFeome, we have constructed the Gateway‐compatible destination vectors pDXTP and pDXTR, which in combination can control the spatial and temporal expression of any open reading frame (ORF). pDXTP receives a promoter/enhancer of interest, which controls the spatial expression of a doxycycline‐inducible transcription factor rtTA. pDXTR receives an ORF of interest, which is controlled by a tetracycline response element enabling temporal control of ORF expression via rtTA activation by simple addition of doxycycline to the rearing water at any desired time point. These vectors can be integrated into the genome via well‐established microinjection‐based SceI, tol2, or phi‐C31 transgenesis procedures and contain fluorescence reporters to confirm transgene integration. Cell‐autonomous verification of ORF expression occurs via red nuclear fluorescence due to an mCherry‐histone H2B fusion protein that is cleaved from the ORF during translation. Function of all essential features of pDXTP and pDXTR has been experimentally validated. pDXTP and pDXTR provide flexible molecular cloning and transgenesis options to accomplish tissue‐specific inducible control of ORF expression in transgenic Xenopus.

中文翻译：

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用于对非洲爪蟾 ORFeome 编码序列进行功能性研究的新型载体。

当前的非洲爪蟾ORFeome 包含约 10,250 个经过验证的全长 cDNA 序列，没有来自非洲爪蟾的终止密码子和约3,970个来自热带非洲爪蟾的终止密码子克隆到与网关兼容的入口向量中。为了增加 ORFeome 的效用，我们构建了网关兼容的目标向量 pDXTP 和 pDXTR，它们结合起来可以控制任何开放阅读框 (ORF) 的空间和时间表达。pDXTP 接收感兴趣的启动子/增强子，它控制多西环素诱导型转录因子 rtTA 的空间表达。pDXTR 接收感兴趣的 ORF，它由四环素响应元件控制，通过在任何所需时间点向饲养水中简单添加强力霉素，通过 rtTA 激活对 ORF 表达进行时间控制。这些载体可以通过成熟的基于显微注射的 SceI、tol2 或 phi-C31 转基因程序整合到基因组中，并包含荧光报告基因以确认转基因整合。由于 mCherry 组蛋白 H2B 融合蛋白在翻译过程中从 ORF 上切割下来，因此通过红色核荧光对 ORF 表达进行细胞自主验证。pDXTP 和 pDXTR 的所有基本特征的功能已经过实验验证。pDXTP 和 pDXTR 提供灵活的分子克隆和转基因选项，以实现对转基因中 ORF 表达的组织特异性诱导控制非洲爪蟾。