Stringent control of gene expression in vivo by using novel doxycycline-dependent trans-activators
Stringent control of gene expression in vivo by using novel doxycycline-dependent trans-activators
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DOI：
10.1089/10430340252769734
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发表时间：
2002-01-01
影响因子：
4.2
通讯作者：
Toniatti, C
中科院分区：
文献类型：
--
作者：
Lamartina, S;Roscilli, G;Toniatti, C
四环素(Tet)依赖的调控系统已被广泛应用于基因表达调控。该系统的Tet-on版本，其中反向Tet-Response转录激活子(RTTA)由Tet或其类似物如强力霉素(Dox)正向调节，在人类基因治疗应用中具有潜在的实用价值。然而，RTTA可能表现出很高的基础活性，特别是当通过使用质粒等异构体载体在体内传递时。已报道了两种新的Dox诱导激活剂rtTA2(S)-S2和rtTA2(S)-M2，它们在稳定表达的细胞系中的基础活性明显低于rtTA。在这项研究中，我们测试了这些反式激活剂控制小鼠促红细胞生成素(MEpo)的表达和调节体内红细胞压积(Hct)增加的能力，通过DNA电注射将质粒导入成年小鼠股四头肌。在体内，rtTA2(S)-M2和rtTA2(S)-S2均表现出比rtTA更低的本底活性和更高的诱导窗口。此外，在没有任何本底活性的情况下，通过注射仅1杯含有rtTA2(S)-S2表达框和Tet反应的mEpo cDNA的单个质粒，在10个月的时间里保持了对mEpo基因表达和Hct水平的严格控制。这是首次报道通过传递一个同时编码反式激活剂和受调控基因的质粒，在体内对基因表达进行严格的配体依赖控制。值得注意的是，基于rtTA2(S)-S2的系统是由口服剂量的多西环素诱导的，与人类临床实践中通常使用的剂量相当。
The tetracycline (Tet)-dependent regulatory system has been widely used for controlling gene expression. The Tet-on version of the system, in which the reverse Tet-responsive transcriptional activator (rtTA) is positively regulated by Tet or its analogs, such as doxycycline (Dox), is of potential utility for gene therapy applications in humans. However, rtTA may display a high basal activity, especially when delivered in vivo by using episomal vectors such as plasmids. Two novel Dox-inducible activators, called rtTA2(S)-S2 and rtTA2(S)-M2, which have a significantly lower basal activity than rtTA in stably transfected cell lines, have been described. In this study we tested the capability of these trans-activators to control expression of mouse erythropoietin (mEpo) and to modulate hematocrit (Hct) increase in vivo on delivery of plasmids into quadriceps muscles of adult mice by DNA electroinjection. Both rtTA2(S)-M2 and rtTA2(S)-S2 displayed a considerably lower background activity and higher window of induction than rtTA in vivo. Moreover, a stringent control of mEpo gene expression and Hct levels in the absence of any background activity was maintained over a 10-month period by injecting as little as 1 mug of a single plasmid containing the rtTA2(S)-S2 expression cassette and the Tet-responsive mEpo cDNA. This constitutes the first report of a stringent ligand-dependent control of gene expression in vivo obtained by delivering a single plasmid encoding both the trans-activator and the regulated gene. Notably, the rtTA2(S)-S2-based system was induced by oral doses of doxycycline comparable to those normally used in clinical practice in humans.