Novel vectors for functional interrogation of Xenopus ORFeome coding sequences.
Novel vectors for functional interrogation of Xenopus ORFeome coding sequences.
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用于非洲爪蟾 ORFeome 编码序列功能询问的新型载体。
DOI：
10.1002/dvg.23329
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发表时间：
2019
期刊：
影响因子：
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通讯作者：
Buchholz,DanielR
中科院分区：
文献类型：
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作者：
Sterner,ZacharyR;Rankin,ScottA;Wlizla,Marcin;Choi,JinyoungA;Luedeke,DavidM;Zorn,AaronM;Buchholz,DanielR
目前的非洲爪蟾ORFeome包含约10，250个来自非洲爪蟾的经验证的全长cDNA序列（不含终止密码子）和约3，970个来自热带爪蟾的全长cDNA序列（克隆到Gateway兼容的入门载体中）。为了增加ORFeome的效用，我们构建了网关兼容的目的载体pDXTP和pDXTR，其组合可以控制任何开放阅读框架（ORF）的空间和时间表达。pDXTP接受感兴趣的启动子/增强子，其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，该ORF由四环素应答元件控制，该四环素应答元件使得能够通过在任何期望的时间点向饲养水中简单地添加多西环素来经由rtTA活化暂时控制ORF表达。这些载体可以通过成熟的基于显微注射的SceI、tol 2或phi-C31转基因程序整合到基因组中，并含有荧光报告基因以确认转基因整合。ORF表达的细胞自主验证通过红色核荧光发生，这是由于mCherry-组蛋白H2 B融合蛋白在翻译过程中从ORF中裂解。pDXTP和pDXTR的所有基本特征的功能已被实验验证。pDXTP和pDXTR提供了灵活的分子克隆和转基因选择，以实现转基因非洲爪蟾中ORF表达的组织特异性诱导控制。
The currentXenopusORFeome contains ~10,250 validated, full‐length cDNA sequences without stop codons fromXenopus laevisand ~3,970 fromXenopus tropicaliscloned 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 transgenicXenopus.