A MODIFIED TETRACYCLINE-REGULATED SYSTEM PROVIDES AUTOREGULATORY, INDUCIBLE GENE-EXPRESSION IN CULTURED-CELLS AND TRANSGENIC MICE
A MODIFIED TETRACYCLINE-REGULATED SYSTEM PROVIDES AUTOREGULATORY, INDUCIBLE GENE-EXPRESSION IN CULTURED-CELLS AND TRANSGENIC MICE
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DOI：
10.1073/pnas.92.14.6522
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发表时间：
1995-07-03
影响因子：
11.1
通讯作者：
SCHATZ, DG
中科院分区：
文献类型：
--
作者：
SHOCKETT, P;DIFILIPPANTONIO, M;SCHATZ, DG
最近描述了一种四环素调节的诱导型基因表达系统，其依赖于四环素控制的反式激活因子（tTA）融合蛋白的组成型表达，该融合蛋白结合了四环素阻遏物和VP 16的转录激活结构域[Gossen，hi，and Bujard，H.等人（1992）Proc. Acad. Sci. USA 89，5547-5551]，该系统仅产生低水平的反式激活蛋白，这可能是因为tTA是有毒的。为了避免这一困难，我们将tTA基因置于tTA结合的诱导型启动子的控制下，使得tTA本身的表达是可诱导的和自动调节的。（RAG-1和RAG-2），自动调节系统产生了显著更高水平的可变（多样性）连接[V（D）J]重组活性（平均70倍）和诱导表达在更大比例的转染细胞（自身调节性，90%，相对于组成性，18%），此外，该系统允许产生转基因小鼠，其中荧光素酶转基因的表达在大多数组织中是基础水平的数十至数百倍经检测，诱导的表达水平在胸腺和肺中最高，并且似乎显著高于先前报道的用组成型系统产生的诱导型荧光素酶转基因小鼠。用经修饰的系统，通过RNA和Western印迹法容易地在细胞系中检测到诱导型反式激活因子mRNA和蛋白，并且通过RNA印迹法在转基因小鼠的一些组织中检测到反式激活因子mRNA，这种自动调节系统代表了培养细胞和转基因动物中四环素调节基因表达的改进策略。
A system for tetracycline-regulated inducible gene expression was described recently which relies on constitutive expression of a tetracycline-controlled transactivator (tTA) fusion protein combining the tetracycline repressor and the transcriptional activation domain of VP16 [Gossen, hi, and Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89, 5547-5551], This system yielded only low levels of transactivator protein, probably because tTA is toxic, To avoid this difficulty, we placed the tTA gene under the control of the inducible promoter to which tTA binds, making expression of tTA itself inducible and autoregulatory, When used to drive expression of the recombination activating genes 1 and 2 (RAG-1 and RAG-2), the autoregulatory system yielded both substantially higher levels of variable (diversity) joining [V(D)J] recombination activity (70-fold on average) and inducible expression in a much larger fraction of transfected cells (autoregulatory, 90%, vs, constitutive, 18%), In addition, this system allowed the creation of transgenic mice in which expression of a luciferase transgene was inducible tens to hundreds of times the basal levels in most tissues examined, Induced levels of expression were highest in thymus and lung and appear to be substantially higher than in previously reported inducible luciferase transgenic mice created with the constitutive system, With the modified system, inducible transactivator mRNA and protein were easily detected in cell lines by RNA and Western blotting, and transactivator mRNA was detected by RNA blotting in some tissues of transgenic mice, This autoregulatory system represents an improved strategy for tetracycline-regulated gene expression both in cultured cells and in transgenic animals.