Efficient transgene regulation from a single tetracycline-controlled positive feedback regulatory system
Efficient transgene regulation from a single tetracycline-controlled positive feedback regulatory system
复制标题
DOI：
10.1038/sj.gt.3300557
复制
发表时间：
1998-01-01
期刊：
影响因子：
5.1
通讯作者：
Hawkins, RE
中科院分区：
文献类型：
--
作者：
A-Mohammadi, S;Hawkins, RE
真核细胞中基因表达的控制在许多应用中显然是重要的，包括治疗性基因产物水平的修饰。为了有效的基因递送和调控，调控系统必须包含在单个载体上，并且它必须在诱导时表现出高的转基因表达和在阻遏时表现出低的基础表达。在这里，我们已经调查了几个独立的载体携带的四环素控制的反式激活因子（tTA）和一个潜在的治疗基因的瞬时研究。无增强子的正反馈调节载体（pSiaIV）从修饰的tTA响应性双向启动子转录tTA和mGM-CSF，在HeLa细胞中表现出超过200倍的基因调节。这与表达tTA和tTA响应性mGM-CSF的载体共转染获得的调节程度相当。pSiaIV的最大转录活性与CMV IE启动子的转录活性相当，并且其基础活性与几种常用细胞系中的四环素响应启动子（tRP）的泄漏一样低，导致47至328倍的调节。此外，pSiaIV在稳定细胞中也显示出有效的调节。正反馈调控系统（PFRS）提供了有效的基因调控，适用于大多数应用，特别是基因治疗。
Control of gene expression in eukaryotic cells is clearly important in many applications including modifications of the level of a therapeutic gene product. For effective gene delivery and regulation, the regulatory system must be contained on a single vector and it must exhibit high transgene expression on induction and low basal expression on repression. Here, we have investigated several self-contained vectors carrying both the tetracycline-controlled transactivator (tTA) and a potentially therapeutic gene in transient studies. An enhancerless positive feedback regulatory vector (pSiaIV) transcribing both tTA and mGM-CSF from a modified tTA-responsive bidirectional promoter demonstrated over 200-fold gene regulation in HeLa cells. This was comparable to the degree of regulation obtained on cotransfection of vectors expressing tTA and tTA-responsive mGM-CSF. The maximal transcriptional activity of pSiaIV was comparable to that of CMV IE promoter and its basal activity as low as the leakiness of the tetracycline-responsive promoter (tRP) in several commonly used cell lines, resulting in 47- to 328-fold regulation. Furthermore, pSiaIV also showed efficient regulation in stable cells. Overall, the positive feedback regulatory system (PFRS) offers efficient gene regulation which is suitable for most applications, especially gene therapy.