An RNA polymerase II construct synthesizes short-hairpin RNA with a quantitative indicator and mediates highly efficient RNAi

Abstract

RNA interference (RNAi) mediates gene silencing in many eukaryotes and has been widely used to investigate gene functions. A common method to induce sustained RNAi is introducing plasmids that synthesize short hairpin RNAs (shRNAs) using Pol III promoters. While these promoters synthesize shRNAs and elicit RNAi efficiently, they lack cell specificity. Monitoring shRNA expression levels in individual cells by Pol III promoters is also difficult. An alternative way to deliver RNAi is to use Pol II-directed synthesis of shRNA. Previous efforts in developing a Pol II system have been sparse and the results were conflicting, and the usefulness of those Pol II vectors has been limited due to low efficacy. Here we demonstrate a new Pol II system that directs efficient shRNA synthesis and mediates strong RNAi at levels that are comparable with the commonly used Pol III systems. In addition, this system synthesizes a marker protein under control of the same promoter as the shRNA, thus providing an unequivocal indicator, not only to the cells that express the shRNA, but also to the levels of the shRNA expression. This system may be adapted for in vivo shRNA expression and gene silencing.

RNA interference against Hec1 inhibits tumor growth in vivo

“…Both vectors carry the same anti-Hec1 sequence: we may speculate that the difference in the protein knockdown could be attributed to different expression of shRNA levels motivated by the respective promoters. Indeed, a recent report 30 shows that the protein knockdown levels by RNAi is higher using H1 or U6 than the CMV promoter. Nevertheless, the authors achieve good repression levels with plasmid constructs carrying a variation of shRNA that mimic human micro-RNA (miR-30) under control of the CMV promoter.…”

The Ste20 Kinases Ste20-related Proline-Alanine-rich Kinase and Oxidative-stress Response 1 Regulate NKCC1 Function in Sensory Neurons

“…As many studies have indicated, shRNA silencing of native genes is generally not as efficient as knockout animals, because complete silencing is rarely obtained (26,27). In our experiments, we chose a short hairpin structure, which adopts the structure of human micro-RNA mir30, and which was reported to produce highly efficient RNAi effect (27).…”

Herpes simplex virus RNAi and neprilysin gene transfer vectors reduce accumulation of Alzheimer's disease-related amyloid-β peptide in vivo

“…Recent literature implies that RNA pol-II promoters can express shRNA efficiently. 33,34 Consequently, we are investigating whether the pol-II-dependent latency active promoter HSV vector-mediated Alzheimer's gene therapy C-S Hong et al LAP2, which has been shown to direct long-term expression of therapeutic transgenes in the central nervous system, 25 can be used either to express shRNA directly in vivo or to prevent inactivation of a chimeric LAP2/pol-III promoter.…”