Stringent doxycycline-dependent control of gene activities using an episomal one-vector system -: art. no. e137
Stringent doxycycline-dependent control of gene activities using an episomal one-vector system -: art. no. e137
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DOI：
10.1093/nar/gni137
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发表时间：
2005-01-01
影响因子：
14.9
通讯作者：
Feuillard, J
中科院分区：
文献类型：
--
作者：
Bornkamm, GW;Berens, C;Feuillard, J
条件表达系统对于剖析复杂的生物现象是至关重要的。在这里，我们描述了一种新的EBV衍生的表体复制质粒(PRTS-1)，它携带了通过Tet调控有条件表达目的基因的所有元件。该载体的特征是(I)低本底活性，(Ii)在多西环素(Dox)存在下的高诱导性和(Iii)对诱导剂浓度增加的分级反应。鸡β肌动蛋白启动子和小鼠免疫球蛋白重链内含子增强子的一个元件驱动编码高度Dox敏感的反向四环素控制的反式激活因子rtTA2(S)-M2的双顺反子表达盒和位于内部核糖体进入位点下游的Tet Repressor-KRAb融合蛋白(TTS(KRAb))(沉默)。目的基因由双向启动子P(Tet)bi-1表达，该双向启动子允许两个基因同时表达，其中一个可用作目的基因表达的替代标记。在没有Dox的情况下，通过将消音剂TTS(KRAb)与P(Tet)bi-1结合来实现严格的下调调节。Dox的加入释放抑制，并通过与rtTA2(S)-M2结合激活P(Tet)bi-1。
Conditional expression systems are of pivotal importance for the dissection of complex biological phenomena. Here, we describe a novel EBV-derived episomally replicating plasmid (pRTS-1) that carries all the elements for conditional expression of a gene of interest via Tet regulation. The vector is characterized by (i) low background activity, (ii) high inducibility in the presence of doxycycline (Dox) and (iii) graded response to increasing concentrations of the inducer. The chicken beta actin promoter and an element of the murine immunoglobin heavy chain intron enhancer drive constitutive expression of a bicistronic expression cassette that encodes the highly Dox-sensitive reverse tetracycline controlled transactivator rtTA2(S)-M2 and a Tet repressor-KRAB fusion protein (tTS(KRAB)) (silencer) placed downstream of an internal ribosomal entry site. The gene of interest is expressed from the bidirectional promoter P(tet)bi-1 that allows simultaneous expression of two genes, of which one may be used as surrogate marker for the expression of the gene of interest. Tight down regulation is achieved through binding of the silencer tTS(KRAB) to P(tet)bi-1 in the absence of Dox. Addition of Dox releases repression and via binding of rtTA2(S)-M2 activates P(tet)bi-1.