Tet-On binary systems for tissue-specific and inducible transgene expression.
Tet-On binary systems for tissue-specific and inducible transgene expression.
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DOI：
10.1007/978-1-61779-992-1_16
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发表时间：
2012-01-01
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通讯作者：
Buchholz, Daniel R
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文献类型：
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作者：
Buchholz, Daniel R
转基因表达的组织特异性和诱导性调控是现代发育生物学研究的基石。尽管这种转基因表达的控制已经在非洲爪蟾中完成，但还没有产生类似于在小鼠和斑马鱼中发现的那些的通用或广泛可用的转基因系组。在这里，我描述了非洲爪蟾中的转基因系的设计和表征，其构成Tet-On二元转基因表达系统，该系统包括两个组分：（1）rtTA转基因系，即，含有在组织特异性启动子控制下的多西环素-（Dox-）依赖性转基因转录因子rtTA的系，和（2）转基因启动子（TRE）转基因系，即，在启动子（TRE）的控制下携带目的基因（下文称为转基因）的品系。在双转基因动物中，即，在含有rtTA和TRE组分的胚胎或蝌蚪中，转基因表达保持不存在Dox。向饲养水中添加Dox引起rtTA的构象变化，允许其结合TRE启动子并诱导转基因表达。转基因表达的组织特异性由调节rtTA表达的启动子决定，诱导性由向饲养水中添加Dox决定。沉积rtTA和TRE转基因系，使组织特异性诱导控制转基因表达到非洲爪蟾股票中心将提供一个强大的和灵活的资源，在发育生物学的研究。
Tissue-specific and inducible control of transgene expression is a cornerstone of modern studies in developmental biology. Even though such control of transgene expression has been accomplished in Xenopus, no general or widely available set of transgenic lines have been produced akin to those found in mouse and zebrafish. Here, I describe the design and characterization of transgenic lines in Xenopus constituting the Tet-On binary transgene expression system comprising two components: (1) rtTA transgenic lines, i.e., lines harboring the doxycycline- (Dox-) dependent transgenic transcription factor rtTA under control of a tissue-specific promoter and (2) transgenic promoter (TRE) transgenic lines, i.e., lines harboring a gene of interest (hereafter called the transgene) under control of a promoter (TRE). In double transgenic animals, i.e., embryos or tadpoles harboring both the rtTA and TRE components, transgene expression remains off the absence of Dox. Addition of Dox to the rearing water causes a conformational change in rtTA allowing it to bind the TRE promoter and induce transgene expression. Tissue specificity of transgene expression is determined by the promoter regulating rtTA expression, and inducibility is determined by the addition of Dox to the rearing water. Deposition of rtTA and TRE transgenic lines enabling tissue-specific inducible control of transgene expression into the Xenopus stock center will provide a powerful and flexible resource for studies in developmental biology.