RNA interference is an effective method to silence specific gene expression. Its application to mammalian cells, however, has been hampered by various shortcomings. Recently, it was reported that introduction of 22-bp double-stranded RNAs (dsRNAs) would specifically suppress expression of endogenous and heterogeneous genes in various mammalian cell lines. However, using this method, we failed to knock out proteins of interest effectively. Here we report the development of a stable and controllable method for generating dsRNA intracellularly. Tetracycline-responsive transactivator-containing cells were transfected with a vector capable of tetracycline-induced bidirectionally overexpressing sense and antisense RNA to form dsRNA in vivo. With this method, glutaredoxin, monitored by Western blot, was knocked out by overexpressing 290-base sense and antisense RNA in NIH 3T3 cells controlled by tetracycline or doxycycline. By using these glutaredoxin knocked-out cells, we have demonstrated that actin deglutathionylation plays a key role in growth factor-mediated actin polymerization, translocalization, and reorganization near the cell periphery.