Cell cycle arrest drastically extends the duration of gene silencing after transient expression of short hairpin RNA.

Targeted gene silencing through RNA interference (RNAi) utilizes short interfering RNA (siRNA) duplexes or vectors expressing short hairpin RNA (shRNA), which is processed in the cells to siRNA. Stable RNAi in mammalian cells is usually achieved through genomic integration of shRNA expressing vectors, but transiently transfected siRNA was also reported to produce long-term silencing in primary mammalian cells. We have developed lentiviral vector LLCEP TU6X for tetracycline/doxycycline-inducible expression of cloned shRNA and a new selectable marker EGFP-Puro, comprising destabilized green fluorescent protein fused with puromycin acetyltransferase. To investigate the stability of gene silencing after transient shRNA expression, LLCEP TU6X vector carrying shRNA against firefly luciferase was transduced into luciferase-expressing human HT1080 fibrosarcoma cells. When doxycycline-induced transcription was followed by the removal of the inducer, EGFP-Puro reverted to basal level within two days, but RNAi activity required six days for full reversion in proliferating cells. When cell division was blocked with mimosine or by inducible expression of cell cycle inhibitors p27 or p21, RNAi effect was undiminished for 4-5 days and maintained at >60% level as late as 21 days after the inducer was removed. In contrast to the phenotypic stability of RNAi, the amount of siRNA in nondividing cells, measured by an RNAse protection assay, decreased approximately 7-fold just one week after transcription shutdown. These results indicate that gene silencing by transiently expressed shRNA is extremely stable in nondividing cells, and that this effect is not merely a consequence of siRNA stability.