Tetracycline-inducible system for regulation of skeletal muscle-specific gene expression in transgenic mice
Tetracycline-inducible system for regulation of skeletal muscle-specific gene expression in transgenic mice
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DOI：
10.1023/a:1022119005836
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发表时间：
2003-02-01
影响因子：
3
通讯作者：
Antin, PB
中科院分区：
文献类型：
--
作者：
Grill, MA;Bales, MA;Antin, PB
对过度表达的严格控制通常是研究基因功能的某一方面或时间点所必需的，并且在转基因中，可能有助于避免普遍存在的过度表达引起的致命效应和并发症。我们利用优化的 tet-on 系统和修饰的肌肉肌酸激酶 (MCK) 启动子的优点，在转基因小鼠中生成骨骼肌特异性、多西环素 (Dox) 控制的过表达系统。生成了 DNA 构建体，其中密码子优化的逆四环素反式激活子 (rtTA) 置于骨骼肌特异性版本的小鼠 MCK 启动子的控制之下。含有该构建体的转基因小鼠几乎只在骨骼肌中表达 rtTA。这些小鼠与第二个转基因品系杂交，该品系含有以驱动荧光素酶报告基因和感兴趣的标记基因的tet响应元件为中心的双向启动子；在这种情况下，钙蛋白酶抑制剂钙蛋白酶抑制剂。复合半合子小鼠表现出高水平、Dox 依赖性肌肉特异性荧光素酶活性，通常比同一小鼠的非肌肉组织高出 10,000 倍。蛋白质印迹和免疫细胞化学分析表明，标记的钙蛋白酶抑制蛋白具有类似的 Dox 依赖性肌肉特异性诱导作用。这些发现证明了 tet-on 系统在成人骨骼肌中提供严格调控的过度表达系统的有效性和灵活性。 MCKrtTA 转基因系可与其他转基因应答系组合，以实现任何感兴趣靶基因的骨骼肌特异性过度表达。
Tightly regulated control of over-expression is often necessary to study one aspect or time point of gene function and, in transgenesis, may help to avoid lethal effects and complications caused by ubiquitous over-expression. We have utilized the benefits of an optimized tet-on system and a modified muscle creatine kinase (MCK) promoter to generate a skeletal muscle-specific, doxycycline (Dox) controlled over-expression system in transgenic mice. A DNA construct was generated in which the codon optimized reverse tetracycline transactivator (rtTA) was placed under control of a skeletal muscle-specific version of the mouse MCK promoter. Transgenic mice containing this construct expressed rtTA almost exclusively in skeletal muscles. These mice were crossed to a second transgenic line containing a bi-directional promoter centered on a tet responder element driving both a luciferase reporter gene and a tagged gene of interest; in this case the calpain inhibitor calpastatin. Compound hemizygous mice showed high level, Dox dependent muscle-specific luciferase activity often exceeding 10,000-fold over non-muscle tissues of the same mouse. Western and immunocytochemical analysis demonstrated similar Dox dependent muscle-specific induction of the tagged calpastatin protein. These findings demonstrate the effectiveness and flexibility of the tet-on system to provide a tightly regulated over-expression system in adult skeletal muscle. The MCKrtTA transgenic lines can be combined with other transgenic responder lines for skeletal muscle-specific over-expression of any target gene of interest.