Matsukura Shiro, Jones Peter A, Takai Daiya
Department of Biochemistry and Molecular Biology, USC/Norris Comprehensive Cancer Center, Keck School of Medicine of the University of Southern California, 1441 Eastlake Avenue, Los Angeles, CA 90033, USA.
Nucleic Acids Res. 2003 Aug 1;31(15):e77. doi: 10.1093/nar/gng077.
Small interference RNA (siRNA) is an emerging methodology in reverse genetics. Here we report the development of a new tetracycline-inducible vector-based siRNA system, which uses a tetracycline-responsive derivative of the U6 promoter and the tetracycline repressor for conditional in vivo transcription of short hairpin RNA. This method prevents potential lethality immediately after transfection of a vector when the targeted gene is indispensable, or the phenotype of the knockdown is lethal or results in a growth abnormality. We show that the controlled knockdown of DNA methyltransferase 1 (DNMT1) in human cancer resulted in growth arrest. Removal of the inducer, doxycycline, from treated cells led to re-expression of the targeted gene. Thus the method allows for a highly controlled approach to gene knockdown.
小干扰RNA(siRNA)是反向遗传学中一种新兴的方法。在此,我们报告了一种基于四环素诱导载体的新型siRNA系统的开发,该系统使用U6启动子的四环素响应衍生物和四环素阻遏物进行短发夹RNA的体内条件性转录。当靶向基因不可或缺,或者敲低的表型是致死性的或导致生长异常时,这种方法可防止在转染载体后立即出现潜在的致死性。我们表明,在人类癌症中对DNA甲基转移酶1(DNMT1)进行可控敲低会导致生长停滞。从处理过的细胞中去除诱导剂强力霉素会导致靶向基因的重新表达。因此,该方法允许对基因敲低进行高度可控的操作。